Expression of ETS related gene (ERG) and phosphatase and tensin homolog (PTEN) correlates with prostate cancer capsular penetration

ABSTRACT

The disclosure provides methods for characterizing a prostate cancer sample by detecting expression of ERG, PTEN or both, changes in which relative to a normal control are shown herein to be correlated with prostate cancer capsular penetration and more aggressive forms of prostate cancer. Such methods are useful for the prognosis of prostate cancer capsular penetration and for making treatment decisions in patients with prostate cancer that has penetrated the capsule. Also provided are kits that can be used with such methods.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/529,691 filed Aug. 31, 2011, herein incorporated by reference in itsentirety.

FIELD

The disclosure provides methods for characterizing a prostate cancersample by detecting changes in expression of ERG and/or PTEN which areshown herein to be correlated with prostate cancer capsular penetration,biochemical recurrence, and more aggressive forms of prostate cancer.Also provided are kits and arrays that can be used with such methods.

BACKGROUND

Oncologists have a number of treatment options available to them,including different combinations of chemotherapeutic drugs that arecharacterized as “standard of care,” and a number of drugs that do notcarry a label claim for particular cancer, but for which there isevidence of efficacy in that cancer. The best chance for a goodtreatment outcome requires that patients promptly receive optimalavailable cancer treatment(s) and that such treatment(s) be initiated asquickly as possible following diagnosis. On the other hand, some cancertreatments have significant adverse effects on quality of life; thus, itis equally important that cancer patients do not unnecessarily receivepotentially harmful and/or ineffective treatment(s).

Prostate cancer provides a good case in point. In 2008, it was estimatedthat prostate cancer alone will account for 25% of all cancers in menand will account for 10% of all cancer deaths in men (Jemal et al., CACancer J. Clin. 58:71-96, 2008). Prostate cancer typically is diagnosedwith a digital rectal exam (“DRE”) and/or prostate specific antigen(PSA) screening. An abnormal finding on DRE and/or an elevated serum PSAlevel (e.g., >4 ng/ml) can indicate the presence of prostate cancer.When a PSA or a DRE test is abnormal, a transrectal ultrasound may beused to map the prostate and show any suspicious areas. Biopsies ofvarious sectors of the prostate are used to determine if prostate canceris present.

The incidence increased with age and the routine availability of serumPSA testing has dramatically increased the number of aging men havingthe diagnosis. In most men the disease is slowly progressive but asignificant number progress to metastatic disease which in time becomesandrogen independent. Prognosis is good if the diagnosis is made whenthe cancer is still localized to the prostate; but nearly one-third ofprostate cancers are diagnosed after the tumor has spread locally, andin 1 of 10 cases, the disease has distant metastases at diagnosis. The5-year survival rate for men with advanced prostate cancer is only33.6%. The choice of appropriate treatment is usually dependent on theage of the patient and the stage of the prostate cancer. This decisionis complicated by the lack of available accurate methods topre-surgically determine the clinical stage and the biologic potentialof a given patient.

An important clinical question is how aggressively to treat suchpatients with prostate cancer. Usual treatment options depend on thestage of the prostate cancer. Men with a 10-year life expectancy or lesswho have a low Gleason number and whose tumor has not spread beyond theprostate often are not treated. Treatment options for more aggressivecancers include radical prostatectomy and/or radiation therapy.Androgen-depletion therapy (such as, gonadotropin-releasing hormoneagonists (e.g., leuprolide, goserelin, etc.) and/or bilateralorchiectomy) is also used, alone or in conjunction with surgery orradiation. However, these prognostic indicators do not accuratelypredict clinical outcome for individual patients. Hence, understandingof the molecular abnormalities that define those tumors at high risk forrelapse is needed to help identify more precise molecular markers.

Nevertheless a few genes have emerged including hepsin (HPN) (Rhodes etal., Cancer Res. 62:4427-33, 2002), alpha-methylacyl-CoA racemase(AMACR) (Rubin et al., JAMA 287:1662-70, 2002), and enhancer of Zestehomolog 2 (EZH2) (Varambally et al., Nature 419:624-9, 2002), which havebeen shown experimentally to have probable roles on prostatecarcinogenesis. Most recently, bioinformatics approaches and geneexpression methods were used to identify fusion of theandrogen-regulated transmembrane protease, serine 2 (TMPRSS2) withmembers of the erythroblast transformation specific (ETS) DNAtranscription factors family (Tomlins et al., Science 310:644-8, 2005).

Another factor impacting clinical utility of the various proposed panelsis the fact that most samples available for validation exist only asformalin fixed paraffin embedded (FFPE) tissues. In contrast, many ofthe cDNA microarray studies conducted to date typically use snap frozentissues (Bibikova et al., Genomics 89:666-72, 2007; van't Veer et al.,Nature 415:530-6, 2002). The ability to perform and analyze geneexpression in FFPE tissues will greatly accelerate research bycorrelating already available clinical information such as histologicalgrade and clinical stage with gene specific signatures.

Given that some prostate cancers need not be treated while others almostalways are fatal and further given that the disease treatment can beunpleasant at best, there is a strong need for methods that allow caregivers to predict the expected course of disease, including thelikelihood of cancer recurrence, long-term survival of the patient, andthe like, and to select the most appropriate treatment optionaccordingly.

SUMMARY

It is disclosed herein that increased ERG expression and decreased PTENexpression (or even absence of detectable PTEN expression) is associatedwith capsular penetration by prostate cancers. For example, detection ofincreased ERG expression and decreased PTEN expression is correlatedwith prostate cancer capsular penetration, and in samples from aprostate cancer that has penetrated the capsule (referred to herein as aCP prostate sample), detection of increased ERG expression and decreasedPTEN expression is indicative of a more aggressive prostate cancer, suchas one likely to biochemically recur. Thus, expression of PTEN and ERG(or even PTEN alone) can be used to forecast prostate cancer outcome(e.g., biochemically recurrence or non-recurrence), for example inpatients who have a prostate cancer that has penetrated the capsule. Inparticular examples, increased ERG expression and decreased PTENexpression indicates an increased likelihood that the prostate cancer ismore aggressive and may recur or metastasize, and thus a poor prognosis.The disclosed methods are useful, for example, to screen prostate cancerpatients for cancer aggressiveness, which can aid prognosis and themaking of therapeutic decisions in prostate cancer. For example,patients identified as having increased ERG expression and decreasedPTEN expression in their prostate cancer sample can be selected for moreaggressive therapies and/or more frequent monitoring, while patientsidentified as not having increased ERG expression and decreased PTENexpression in their prostate cancer sample can be selected for lessaggressive therapies and/or less frequent monitoring. Methods andcompositions (including kits and arrays that include antibodies orprobes that specifically bind to ERG and PTEN) that embody thisdiscovery are described.

Methods are provided for characterizing a prostate cancer. Such methodscan include detecting or measuring expression of ERG, PTEN, or both in aprostate cancer sample from a subject, such as a CP prostate cancersample. The expression of ERG and PTEN in the prostate cancer sample iscompared to a control representing ERG and PTEN expression expected in anormal prostate sample (e.g., ERG− and PTEN+) and diagnosing orprognosing that the prostate cancer is more aggressive when increasedexpression of ERG and decreased expression of PTEN is detected in theprostate cancer sample relative to the control. For example, a moreaggressive prostate cancer can be one which is more likely tobiochemically recur, metastasize, less likely to respond to treatment,or combinations thereof, such as an increased likelihood that theprostate cancer will biochemically recur following prostatectomy within1, 3, or 5 years.

In addition, kits that include one or more means for detecting in abiological sample an ERG genomic sequence, ERG transcript ERG protein,PTEN genomic sequence, PTEN transcript or PTEN protein, or anycombination of any of the foregoing, are provided.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the types of prostate tumors analyzed, thosehaving capsular penetration (CP) and those that do not invade theprostatic capsule (NCP).

FIG. 2 is a digital image showing staining of prostate carcinoma withH&E (left) an ERG-specific antibody (right).

FIG. 3 is a digital image showing a prostate carcinoma from a case withcapsule penetration and no detectable PTEN (left) and ERG-specificstaining (right).

FIG. 4 is a digital image showing a prostate carcinoma with capsulepenetration and detectable PTEN (left) and ERG-specific staining(right).

FIG. 5 is a digital image showing heterogeneous ERG expression in aprostate cancer that has not penetrated the capsule.

FIG. 6 is a digital image showing IHC results for ERG (left) and PTEN(right) expression in a prostate cancer sample.

FIG. 7 is a graph showing that ERG+ and PTEN− prostate cancers are veryhighly aggressive, as indicated by early biochemical recurrence.

FIG. 8 is a schematic drawing showing the quantum dot FISH detectionplatform used to detect ERG and PTEN genomic DNA.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. All sequence database accession numbers referencedherein are understood to refer to the version of the sequence identifiedby that accession number as it was available on the filing date of thisapplication. In the accompanying sequence listing:

SEQ ID NOS: 1 and 2 are a human Ets related gene (ERG) nucleic acidcoding sequence and corresponding protein sequence.

SEQ ID NOS: 3 and 4 are a human phosphatase and tensin homolog (PTEN)nucleic acid coding sequence and corresponding protein sequence.

SEQ ID NOS: 5 and 6 are primers that can be used to amplify ERG.

SEQ ID NOS: 7 and 8 are primers that can be used to amplify ERG.

SEQ ID NOS: 9 and 10 are primers that can be used to amplify PTEN.

DETAILED DESCRIPTION I. Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which a disclosed invention belongs. Definitions ofcommon terms in molecular biology may be found in Benjamin Lewin, GenesV, published by Oxford University Press, 1994 (ISBN 0-19-854287-9);Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. “Comprising” means “including”; hence, “comprising A or B”means “including A” or “including B” or “including A and B.”

Suitable methods and materials for the practice and/or testing ofembodiments of the disclosed methods are described below. Such methodsand materials are illustrative only and are not intended to be limiting.Other methods and materials similar or equivalent to those describedherein also can be used. For example, conventional methods well known inthe art to which a disclosed invention pertains are described in variousgeneral and more specific references, including, for example, Sambrooket al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel etal., Current Protocols in Molecular Biology, Greene PublishingAssociates, 1992 (and Supplements to 2000); Ausubel et al., ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1999.

All sequences associated with the GenBank® accession numbers referencedherein are incorporated by reference (e.g., the sequence present on Aug.31, 2011 is incorporated by reference).

In order to facilitate review of the various disclosed embodiments, thefollowing explanations of specific terms are provided:

Amplification of a Nucleic Acid Molecule:

Refers to methods used to increase the number of copies of a nucleicacid molecule, such as an ERG or PTEN nucleic acid molecule. Theresulting products can be referred to as amplicons or amplificationproducts. Methods of amplifying nucleic acid molecules are known in theart, and include MDA, PCR (such as RT-PCR and qRT-PCR), DOP-PCR, RCA,T7/Primase-dependent amplification, SDA, 3SR, NASBA, and LAMP, amongothers.

Antibody:

A polypeptide ligand including at least a light chain or heavy chainimmunoglobulin variable region which specifically recognizes and bindsan epitope of an antigen, such as an endothelial marker or a fragmentthereof. Antibodies are composed of a heavy and a light chain, each ofwhich has a variable region, termed the variable heavy (V_(H)) regionand the variable light (V_(L)) region. Together, the V_(H) region andthe V_(L) region are responsible for binding the antigen recognized bythe antibody. In one example, an antibody specifically binds to an ERGor PTEN protein, but not other proteins (such as other proteins found inhuman prostate tissue or that are associated with prostate cancer suchas prostate-specific antigen, PSA). Thus, the disclosure providesantibodies that specifically bind to an ERG or PTEN protein, such as amonoclonal or polyclonal antibody specific for ERG or PTEN, such as alabeled monoclonal or polyclonal antibody.

This includes intact immunoglobulins and the variants and portions ofthem well known in the art, such as Fab′ fragments, F(ab)′₂ fragments,single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies (for example, humanized murineantibodies), heteroconjugate antibodies (such as, bispecificantibodies). See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed., W.H.Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs”. The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The Kabat database is now maintained online. The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDRs inthree-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. For example, an antibody that binds ERG or PTENwill have a specific V_(H) region and the V_(L) region sequence, andthus specific CDR sequences. Antibodies with different specificities(i.e. different combining sites for different antigens) have differentCDRs. Although it is the CDRs that vary from antibody to antibody, onlya limited number of amino acid positions within the CDRs are directlyinvolved in antigen binding. These positions within the CDRs are calledspecificity determining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “polyclonal antibody” is an antibody that is derived from differentB-cell lines. Polyclonal antibodies are a mixture of immunoglobulinmolecules secreted against a specific antigen, each recognizing adifferent epitope. These antibodies are produced by methods known tothose of skill in the art, for instance, by injection of an antigen intoa suitable mammal (such as a mouse, rabbit or goat) that induces theB-lymphocytes to produce IgG immunoglobulins specific for the antigenwhich are then purified from the mammal's serum.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds anendothelial marker.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (for example amouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one embodiment, allthe CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they are substantially identical to human immunoglobulin constantregions, e.g., at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human immunoglobulin sequences. Humanized immunoglobulins can beconstructed by means of genetic engineering (see for example, U.S. Pat.No. 5,585,089).

Binding Affinity:

Affinity of one molecule for another, such as an antibody for an antigen(for example, an ERG or PTEN protein). In one example, affinity iscalculated by a modification of the Scatchard method described byFrankel et al., Mol. Immunol., 16:101-106, 1979. In another example,binding affinity is measured by an antigen/antibody dissociation rate.In yet another example, a high binding affinity is measured by acompetition radioimmunoassay. In several examples, a high bindingaffinity is at least about 1×10⁻⁸M. In other examples, a high bindingaffinity is at least about 1.5×10⁻⁸, at least about 2.0×10⁻⁸, at leastabout 2.5×10⁻⁸, at least about 3.0×10⁻⁸, at least about 3.5×10⁻⁸, atleast about 4.0×10⁻⁸, at least about 4.5×10⁻⁸, or at least about5.0×10⁻⁸ M.

Capsular Penetration or Invasion:

When referring to a prostate cancer, refers to when prostate cancerextends into and in some examples through the prostatic capsule.

Cancer:

Malignant neoplasm, for example one that has undergone characteristicanaplasia with loss of differentiation, increased rate of growth,invasion of surrounding tissue, and is capable of metastasis.

Complementary:

A nucleic acid molecule is said to be “complementary” with anothernucleic acid molecule if the two molecules share a sufficient number ofcomplementary nucleotides to form a stable duplex or triplex when thestrands bind (hybridize) to each other, for example by formingWatson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable bindingoccurs when a nucleic acid molecule (e.g., nucleic acid probe or primer)remains detectably bound to a target nucleic acid sequence (e.g., ERG orPTEN target nucleic acid sequence) under the required conditions.

Complementarity is the degree to which bases in one nucleic acidmolecule (e.g., nucleic acid probe or primer) base pair with the basesin a second nucleic acid molecule (e.g., target nucleic acid sequence,such as ERG or PTEN). Complementarity is conveniently described bypercentage, that is, the proportion of nucleotides that form base pairsbetween two molecules or within a specific region or domain of twomolecules. For example, if 10 nucleotides of a 15 contiguous nucleotideregion of a nucleic acid probe or primer form base pairs with a targetnucleic acid molecule, that region of the probe or primer is said tohave 66.67% complementarity to the target nucleic acid molecule.

In the present disclosure, “sufficient complementarity” means that asufficient number of base pairs exist between one nucleic acid moleculeor region thereof (such as a region of a probe or primer) and a targetnucleic acid sequence (e.g., a ERG or PTEN nucleic acid sequence) toachieve detectable binding. A thorough treatment of the qualitative andquantitative considerations involved in establishing binding conditionsis provided by Beltz et al. Methods Enzymol. 100:266-285, 1983, and bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989.

Contact:

To bring one agent into close proximity to another agent, therebypermitting the agents to interact. For example, an antibody (or otherspecific binding agent) can be applied to a microscope slide or othersurface containing a biological sample, thereby permitting detection ofproteins (or protein-protein interactions or protein-nucleic acidinteractions) in the sample that are specific for the antibody. Inanother example, a oligonucleotide probe or primer (or other nucleicacid binding agent) can be incubated with nucleic acid moleculesobtained from a biological sample (and in some examples under conditionsthat permit amplification of the nucleic acid molecule), therebypermitting detection of nucleic acid molecules (or nucleic acid-nucleicacid interactions) in the sample that have sufficient complementarity tothe probe or primer.

Control:

A sample or standard used for comparison with a test sample, such as abiological sample, e.g., a biological sample obtained from a patient (orplurality of patients) or a cell culture. In some embodiments, thecontrol is a sample obtained from a healthy patient (or plurality ofpatients) (also referred to herein as a “normal” control), such as anormal sample (e.g., one that does not have prostate cancer, such as anormal prostate sample). In some embodiments, the control is ahistorical control or standard value (i.e., a previously tested controlsample or group of samples that represent baseline or normal values). Insome embodiments the control is a standard value representing theaverage value (or average range of values) obtained from a plurality ofpatient samples. In some examples, the control is ERG expression inendothelial cells and/or PTEN expression in a normal prostate gland.

A control can also be represented by a reference value or range ofvalues representing an amount of activity or expression determined to berepresentative of a given condition. Reference values can include arange of values, real or relative expected to occur under certainconditions. These values can be compared with experimental values todetermine if a given molecule is up-regulated or down-regulated in aparticular sample for instance. In one example, a reference value orrange of values represents an amount of activity or expression of ERGand/or PTEN proteins and/or nucleic acids in a sample, such as a samplefrom a subject without prostate cancer (such as a healthy male prostatesample), or non-cancerous tissue adjacent to the prostate cancer in fromthe same or other patients. This value can then be used to determine ifthe subject from whom a test sample was obtained has a more aggressiveform of prostate cancer by comparing this reference value of expressionto the level of expression detected in the test sample. In a particularexample, a decrease in PTEN and an increase in ERG expression oractivity in a test sample as compared to a reference value for anERG−/PTEN+ prostate sample indicates that the subject has a moreaggressive prostate cancer.

Detect:

To determine if an agent (e.g., a nucleic acid molecule or protein) orinteraction (e.g., binding between two proteins, between a protein and anucleic acid, or between two nucleic acid molecules) is present orabsent in a sample, for example by making measurements from a sample. Insome examples this can further include quantification. In particularexamples, an emission signal from a label is detected. Detection can bein bulk, so that a macroscopic number of molecules can be observedsimultaneously. Detection can also include identification of signalsfrom single molecules using microscopy and such techniques as totalinternal reflection to reduce background noise.

For example, use of an antibody specific for a particular protein (e.g.,ERG or PTEN) permits detection of the of the protein or protein-proteininteraction in a sample, such as a sample containing prostate cancertissue. In another example, use of a probe or primer specific for aparticular gene (e.g., ERG or PTEN) permits detection of the desirednucleic acid molecule in a sample, such as a sample containing prostatecancer tissue.

Diagnose:

The process of identifying a medical condition or disease, for examplefrom the results of one or more diagnostic procedures. In one example,the disclosed methods allow for diagnosis of a prostate cancer that haspenetrated the prostatic capsule if elevated ERG and decreased PTEN isdetected (as compared to expected levels of ERG and PTEN in a normalsample).

Differential Expression:

A nucleic acid sequence is differentially expressed when the amount ofone or more of its expression products (e.g., transcript (e.g., mRNA)and/or protein) is higher or lower in one tissue (or cell) type ascompared to another tissue (or cell) type. Detecting differentialexpression can include measuring a change in gene or protein expression,such as a change in ERG and PTEN expression. For example, a gene, e.g.,ERG, the transcript or protein of which is more highly expressed in amore aggressive prostate cancer tissue (or cells) that has penetratedthe prostatic capsule and less expressed in a less aggressive prostatecancer tissue (or cells) that has penetrated the prostatic capsule, isdifferentially expressed. In another example, a gene, e.g., PTEN, thetranscript or protein of which is less expressed (or even not expressed)in a more aggressive prostate cancer tissue (or cells) that haspenetrated the prostatic capsule and more greatly expressed in a lessaggressive prostate cancer tissue (or cells) that has penetrated theprostatic capsule, is differentially expressed.

Downregulated or Inactivation:

When used in reference to the expression of a nucleic acid molecule(such as PTEN), such as a gene, refers to any process which results in adecrease or elimination in production of a gene product, such as a PTENprotein. A gene product can be RNA (such as mRNA, rRNA, tRNA, andstructural RNA) or protein. Therefore, downregulation or deactivationincludes processes that decrease or even eliminate transcription of agene or translation of mRNA.

Examples of processes that decrease transcription include those thatfacilitate degradation of a transcription initiation complex, those thatdecrease transcription initiation rate, those that decreasetranscription elongation rate, those that decrease processivity oftranscription and those that increase transcriptional repression. Genedownregulation can include reduction of expression above an existinglevel. Examples of processes that decrease translation include thosethat decrease translational initiation, those that decreasetranslational elongation and those that decrease mRNA stability.

Downregulation includes any detectable decrease in the production of agene product. In certain examples, production of a gene productdecreases by at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, at least 10-fold, or at least 20-fold (or even not detectable)as compared to a control (such an amount of protein or nucleic acidexpression detected in a normal prostate cell). For example PTEN nucleicacids and proteins are more likely downregulated in subjects who haveaggressive forms of prostate cancer with capsular penetration, such as aprostate cancer likely to biochemically recur following prostatectomy.In one example, a control is a relative amount of gene expression orprotein expression in a prostate sample from a subject (or population ofsubjects) who does not have prostate cancer.

Expression:

The process by which the coded information of a gene is converted intoan operational, non-operational, or structural part of a cell, such asthe synthesis of a protein. Gene expression can be influenced byexternal signals (such as a hormone). Expression of a gene also can beregulated anywhere in the pathway from DNA to RNA to protein. Regulationcan include controls on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization or degradation ofspecific protein molecules after they are produced.

The expression of an ERG or PTEN nucleic acid molecule or protein can bealtered relative to a normal (wild type) nucleic acid molecule orprotein (such as in a patient not having prostate cancer or has aprostate cancer that has not penetrated the capsule). Alterations ingene expression, such as differential expression, include but are notlimited to: (1) overexpression (e.g., upregulation); (2) underexpression(e.g., downregulation); or (3) suppression of expression. Alternationsin the expression of a nucleic acid molecule can be associated with, andin fact cause, a change in expression of the corresponding protein.

Protein expression can also be altered in some manner to be differentfrom the expression of the protein in a normal (wild type) situation.This includes but is not necessarily limited to: (1) a mutation in theprotein such that one or more of the amino acid residues is different;(2) a short deletion or addition of one or a few (such as no more than10-20) amino acid residues to the sequence of the protein; (3) a longerdeletion or addition of amino acid residues (such as at least 20residues), such that an entire protein domain or sub-domain is removedor added; (4) expression of an increased amount of the protein comparedto a control or standard amount (e.g., upregulation); (5) expression ofa decreased amount of the protein compared to a control or standardamount (e.g., downregulation); (6) alteration of the subcellularlocalization or targeting of the protein; (7) alteration of thetemporally regulated expression of the protein (such that the protein isexpressed when it normally would not be, or alternatively is notexpressed when it normally would be); (8) alteration in stability of aprotein through increased longevity in the time that the protein remainslocalized in a cell; and (9) alteration of the localized (such as organor tissue specific or subcellular localization) expression of theprotein (such that the protein is not expressed where it would normallybe expressed or is expressed where it normally would not be expressed),each compared to a control or standard. Controls or standards forcomparison to a sample, for the determination of differentialexpression, include samples believed to be normal (in that they are notaltered for the desired characteristic, for example a sample from asubject who does not have prostate cancer) as well as laboratory values,even though possibly arbitrarily set, keeping in mind that such valuescan vary from laboratory to laboratory.

Laboratory standards and values may be set based on a known ordetermined population value and can be supplied in the format of a graphor table that permits comparison of measured, experimentally determinedvalues.

Gene:

A nucleic acid (e.g., genomic DNA, cDNA, or RNA) sequence that comprisescoding sequences necessary for the production of a polypeptide,precursor, or RNA (e.g., mRNA). The polypeptide can be encoded by afull-length coding sequence or by any portion of the coding sequence solong as the desired activity or functional properties (e.g., enzymaticactivity, ligand binding, signal transduction, immunogenicity, etc.) ofthe full-length or fragment is/are retained. The term also encompassesthe coding region of a structural gene and the sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof about 1 kb or more on either end such that the gene corresponds tothe full-length mRNA. Sequences located 5′ of the coding region andpresent on the mRNA are referred to as 5′ untranslated sequences.Sequences located 3′ or downstream of the coding region and present onthe mRNA are referred to as 3′ untranslated sequences. The gene aspresent in (or isolated from) a genome contains the coding regions(“exons”) interrupted with non-coding sequences termed “introns.”Introns are absent in the processed RNA (e.g., mRNA) transcript.

Label:

An agent capable of detection, for example by spectrophotometry, flowcytometry, or microscopy. For example, one or more labels can beattached to an antibody, thereby permitting detection of a targetprotein (such as ERG or PTEN). Furthermore, one or more labels can beattached to a nucleic acid molecule, thereby permitting detection of atarget nucleic acid molecule (such as ERG or PTEN DNA or RNA). Exemplarylabels include radioactive isotopes, fluorophores, chromophores,ligands, chemiluminescent agents, enzymes, and combinations thereof.

Normal Cells or Tissue:

Non-tumor, non-malignant cells and tissue.

Prognose:

The process of determining the likely outcome of a subject having adisease (e.g., prostate cancer) in the absence of additional therapy. Inone example, the disclosed methods allow for prognosis of future events,such as the likely biochemical recurrence of prostate cancer afterprostatectomy (e.g., likelihood of biochemical recurrence in 1 year, 3years or 5 years) after pro statectomy, and/or predicting the likelymetastasis of a prostate cancer (e.g., after prostatectomy).

Specific Binding (or Derivations of Such Phrase, Such as SpecificallyBinds, Specific for, Etc.):

The particular interaction between one binding partner (such as agene-specific probe or protein-specific antibody) and another bindingpartner (such as a target of a gene-specific probe or protein-specificantibody). Such interaction is mediated by one or, typically, morenon-covalent bonds between the binding partners (or, often, between aspecific region or portion of each binding partner). In contrast tonon-specific binding sites, specific binding sites are saturable.Accordingly, one exemplary way to characterize specific binding is by aspecific binding curve. A specific binding curve shows, for example, theamount of one binding partner (the first binding partner) bound to afixed amount of the other binding partner as a function of the firstbinding partner concentration. As the first binding partnerconcentration increases under these conditions, the amount of the firstbinding partner bound will saturate. In another contrast to non-specificbinding sites, specific binding partners involved in a directassociation with each other (e.g., a probe-mRNA or antibody-proteininteraction) can be competitively removed (or displaced) from suchassociation by excess amounts of either specific binding partner. Suchcompetition assays (or displacement assays) are very well known in theart.

Subject:

Includes any multi-cellular vertebrate organism, such as human andnon-human mammals (e.g., veterinary subjects). In some examples, asubject is one who has cancer, or is suspected of having cancer, such asprostate cancer, such as a prostate cancer with capsular penetration.

Upregulated or Activation:

When used in reference to the expression of a molecule, such as a geneor protein, refers to any process which results in an increase inproduction of a gene product. A gene product can be RNA (such as mRNA,rRNA, tRNA, and structural RNA) or protein. Therefore, upregulation oractivation includes processes that increase the presence of ERG proteinsor nucleic acids, for example fusions between ERG and TMPRSS resultingin TMPRSS/ERG rearrangements.

Examples of processes that increase transcription include those thatfacilitate formation of a transcription initiation complex, those thatincrease transcription initiation rate, those that increasetranscription elongation rate, those that increase processivity oftranscription and those that relieve transcriptional repression (forexample by blocking the binding of a transcriptional repressor).Upregulation can include inhibition of repression as well as stimulationof expression above an existing level. Examples of processes thatincrease translation include those that increase translationalinitiation, those that increase translational elongation and those thatincrease mRNA stability.

Upregulation includes any detectable increase in the production of agene product, such as an ERG protein. In certain examples, detectableERG protein or nucleic acid expression in an aggressive prostate cancerthat has penetrated the capsule increases by at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, or at least 10-fold ascompared to a control (such an amount of detectable ERG protein ornucleic acid in a normal sample). For example ERG is upregulated insubjects who have or a more likely to have an aggressive prostate cancerthat has penetrated the capsule. In one example, a control is a relativeamount of expression in a normal sample.

II. ERG and PTEN Expression Correlate with Capsular Penetration inProstate Cancer

It is disclosed herein that increased expression of ERG and decreased oreven eliminated expression of PTEN, relative to such expression innormal prostate tissues (e.g., an ERG−/PTEN+ prostate sample), arepredictive of increased risk of capsular penetration in prostatecancers, more aggressive prostate cancer, increased likelihood ofbiochemical recurrence, and poor prognosis. In some examples, decreasedor even eliminated expression of PTEN, relative to such expression innormal prostate tissues (e.g., an ERG−/PTEN+ prostate sample), arepredictive of increased likelihood of prostate cancer biochemicalrecurrence following prostatectomy. Methods and compositions that embodythis discovery are described.

Provided herein are methods of characterizing a prostate cancer, such asa prostate cancer that has penetrated the prostatic capsule. Inparticular examples, the method is diagnostic, in that it determinesthat a prostate cancer has penetrated the prostatic capsule. Inparticular examples, the method is prognostic, in that it predicts thelikelihood that the prostate cancer will penetrate the prostaticcapsule. In other examples, the method is prognostic, in that itpredicts whether the prostate cancer is likely to or biochemically recur(for example after prostatectomy). Biochemical recurrence (BCR) is anydetectable prostate specific antigen (PSA) after prostatectomy or a PSArise after a period of PSA detection absence (see Simmons et al., Eur.Urol. 51:1175-84, 2007). After prostatectomy, PSA levels typicallydecrease to an undetectable level after about four weeks. In someexamples, BCR is indicated by PSA levels ≧0.2 ng/ml, such as ≧0.4 ng/mlor ≧0.2 to 0.6 ng/ml, such as two successive PDS levels ≧0.2 ng/ml or≧0.4 ng/ml, following prostatectomy. BCR is indicative of the presenceof prostatic epithelial tissue, and is assumed to represent cancer.

The sample can be a fixed, wax-embedded prostate cancer tissue sample,such as a fixed, wax-embedded prostate cancer tissue sample, which hasor has not penetrated the capsule. In some examples, the prostate cancersample is collected after prostate cancer diagnosis and afterprostatectomy in the subject, or can be collected during aprostatectomy. In some examples, the prostate cancer sample is aprostate cancer that has penetrated the prostatic capsule. In otherexamples, the sample is obtained during a prostate tissue biopsy of atumor suspected or known to be cancerous or suspected or known to havepenetrated the capsule.

In particular methods, the method includes detecting or measuringexpression of ERG, PTEN, or both in a prostate cancer sample from asubject (for example determining ERG and PTEN expression levelsqualitatively or quantitatively). The expression of ERG and PTEN can bedetected at the genomic level, or gene expression products can bedetected, such as ERG and PTEN nucleic acids (e.g., mRNA, cDNA) orproteins. In some examples, determining the expression level includesdetecting alteration(s) in the genomic sequence(s) of ERG or PTEN, suchas detecting a TMPRSS-ERG rearrangement or PTEN deletion. For example,the method can include detecting amplification of at least one ERGallele, fusion of at least one ERG allele, deletion of at least one PTENallele, or combinations thereof.

The ERG or PTEN expression, or both, in the test prostate cancer sampleare compared to ERG and PTEN expression in a control, such as a controlthat represents ERG and PTEN expression expected in a normal prostatesample (which are ERG− and PTEN+) or normal endothelial cells. Based onthe expression levels of ERG and PTEN in the test sample as compared tothe control, the prostate cancer is characterized, such as diagnosed orprognosed. In some examples, the method can include one or more ofobtaining the prostate cancer sample (e.g., a sample from a prostatecancer that has penetrated the capsule); fixing the sample; andcontacting the sample with ERG and PTEN specific binding agents (e.g.,nucleic acid probes or antibodies).

For example, the method can include determining that there is a higherlikelihood that the prostate cancer will penetrate the prostatic capsulein the future when increased expression of ERG and decreased expressionof PTEN is detected in the prostate cancer sample relative to the normalcontrol. In another example, the method can include determining ordiagnosing that the prostate cancer has penetrated the prostatic capsulewhen increased expression of ERG and decreased expression of PTEN isdetected in the prostate cancer sample relative to the normal control.In another example, the method can diagnose that the prostate cancerthat has penetrated the prostatic capsule is more aggressive whenincreased expression of ERG and decreased expression of PTEN is detectedin the prostate cancer sample relative to the control. In anotherexample, the method can prognose an increased likelihood that theprostate cancer that has penetrated the prostatic capsule is moreaggressive when increased expression of ERG and decreased expression ofPTEN is detected in the prostate cancer sample relative to the control.In another example, the method can prognose an increased likelihood thatthe prostate cancer will biochemically recur when decreased expressionof PTEN (in some examples in combination with increased expression ofERG) is detected in the prostate cancer sample relative to the control.

In one example, determining that there is a higher or greater likelihoodthat the prostate cancer will penetrate the prostatic capsule in thefuture when increased expression of ERG and decreased expression of PTENis detected in the prostate cancer sample relative to the normal control(e.g., an ERG−/PTEN+ prostate sample), indicates that the prostatecancer is about 3 times to about 5 times more likely (such as about 4times to about 5 times, about 4.5 times to about 5 times, or about 3, 4,4.5, 4.8, 4.9, or 5 times, more likely) to penetrate the prostaticcapsule, than a prostate cancer sample that does not have increasedexpression of ERG and decreased expression of PTEN relative to thecontrol.

In one example, a prognosis that the cancer is more aggressive indicatesthat the prostate cancer is predicted to biochemically recur within 1year, within 3 years or within 5 years of a prostatectomy. In oneexample, a prognosis that the cancer is more aggressive indicates thatthe prostate cancer is predicted to metastasize within 1 year, within 3years or within 5 years of a prostatectomy. For example, a prognosisthat the cancer is more likely to biochemically recur or metastasize canbe relative to a prostate cancer sample that does not have increasedexpression of ERG and decreased expression of PTEN relative to thecontrol (e.g., an ERG−/PTEN+ prostate sample).

In one example, a prognosis that the cancer is less aggressive (e.g.,levels of ERG ad PTEN similar to a normal prostate; that is ERG notsignificantly upregulated and significant PTEN expression detected)indicates that the prostate cancer is predicted to not biochemicallyrecur within 1 year, within 3 years or within 5 years of aprostatectomy. In one example, a diagnosis or prognosis that the canceris less aggressive indicates that the prostate cancer is not predictedto metastasize within 1 year, within 3 years or within 5 years of aprostatectomy.

The method can further include detecting or measuring expression of oneor more other prostate cancer related molecules in the sample andcomparing expression of the one or more other prostate cancer relatedmolecules in the prostate cancer sample to a control representingexpression of the one or more other prostate cancer related moleculesexpected in a normal prostate sample. Prostate cancer related moleculesinclude those whose expression is known to be altered (such as increasedor decreased) in a prostate cancer sample, relative to a normal prostatecancer sample. Examples include but are not limited to: growtharrest-specific 1 (GAS 1; OMIM 139185), wingless-type MMTV integrationsite family member 5 (WNT5A; OMIM 164975), thymidine kinase 1 (TK1; OMIM188300), V-raf murine sarcoma viral oncogene homolog B1 (BRAF; OMIM164757), ETS translocation variant 4 (ETV4; OMIM 600711), tumor proteinp63 (OMIM 603273), BCL-2 (OMIM 151430), Ki67 (OMIM 176741), ERK5 (OMIM602521), prostate specific antigen (PSA; OMIM 176820), ETS translocationvariant 1 (ETV1; OMIM 600541), measures of nuclear morphology (includingnuclear size and shape variation characteristics), or combinationsthereof.

In some examples, the method can further include detecting expression ofone or more control molecules in the sample and comparing expression ofthe one or more control molecules in the prostate cancer sample to acontrol representing expression of the one or more control moleculesexpected in a normal prostate sample. In some examples, expression ofERG and/or PTEN is normalized to expression of one or more internalcontrols, such as ERG expression in endothelial cells or PTEN expressionin a normal gland.

A. Methods of Use

This disclosure identifies ERG and PTEN are differentially expressed inprostate cancer that has penetrated the capsule and more aggressiveprostate cancers that have penetrated the capsule. In addition, it isshown that PTEN is down-regulated in prostate cancer that is more likelyto biochemically recur following prostatectomy. A more-aggressiveprostate cancer can be indicated by recurrence of after treatment (e.g.,prostatectomy), a worse prognosis for the patient (e.g., decreasedsurvival time), an increased likelihood of disease progression (e.g.,metastasis), failure (or inadequacy) of treatment, and/or a need foralternative (or additional) treatments. Accordingly, the presentdiscoveries have enabled, among other things, a variety of methods forcharacterizing prostate cancer tissues, diagnosis or prognosis ofprostate cancer patients, predicting treatment outcome in prostatecancer patients, and directing (e.g., selecting useful) treatmentmodalities for prostate cancer patients (such as a patient with aprostate cancer that has penetrated the capsule).

In some examples, the disclosed methods can be used to identify thosesubjects that will benefit from a more or less aggressive therapy. Forexample, if a patient is diagnosed or prognosed with an aggressive formof prostate cancer, the patient can be selected for more aggressivetreatment and frequent monitoring. In contrast, if a patient isdiagnosed or prognosed with a less aggressive form of prostate cancer,the patient can be selected for less aggressive treatment and lessfrequent monitoring. Thus in some examples, the disclosed methodsfurther include selecting a patient for more or less aggressivetreatment or monitoring, depending on the ERG and PTEN expressiondetected. For example, such diagnostic or prognostic methods can beperformed prior to the subject undergoing the treatment. Thus, themethods of the present disclosure are valuable tools for practicingphysicians to make quick treatment decisions regarding how to treatprostate cancer, such as prostate cancer that has penetrated thecapsule. These treatment decisions can include the administration of anagent for treating prostate cancer and decisions to monitor a subjectfor recurrence or metastasis of a prostate cancer that has penetratedthe capsule. Thus, in some examples, the disclosed methods furtherinclude treating the subject with prostate cancer, for exampleadministering one or more therapeutic agents for treating prostatecancer, such as chemotherapeutics (e.g., temozolomide or docetaxel) orhormone therapy, treating the patient with radiation therapy (e.g.,prostate brachytherapy), performing a prostatectomy, or combinationsthereof.

Disclosed methods can be performed using biological samples obtainedfrom a subject having prostate cancer, such as a prostate cancer thathas or has not invaded the prostatic capsule. A typical subject is ahuman male; however, any mammal that has a prostate that may developprostate cancer can serve as a source of a biological sample useful in adisclosed method. Exemplary biological samples useful in a disclosedmethod include tissue samples (such as prostate biopsies and/orprostatectomy tissues) or prostate cell samples (such as can becollected by prostate massage, in the urine, or in fine needleaspirates). In one example, the sample is a prostate cancer sample thathas penetrated the capsular region. Samples may be fresh or processedpost-collection (e.g., for archiving purposes). In some examples,processed samples may be fixed (e.g., formalin-fixed) and/or wax- (e.g.,paraffin-) embedded. Fixatives for mounted cell and tissue preparationsare well known in the art and include, without limitation, 95% alcoholicBouin's fixative; 95% alcohol fixative; B5 fixative, Bouin's fixative,formalin fixative, Karnovsky's fixative (glutaraldehyde), Hartman'sfixative, Hollande's fixative, Orth's solution (dichromate fixative),and Zenker's fixative (see, e.g., Carson, Histotechology: ASelf-Instructional Text, Chicago:ASCP Press, 1997). Particular methodembodiments involve FFPE prostate cancer tissue samples. In someexamples, the sample (or a fraction thereof) is present on a solidsupport. Solid supports useful in a disclosed method need only bear thebiological sample and, optionally, but advantageously, permit theconvenient detection of components (e.g., proteins and/or nucleic acidsequences) in the sample. Exemplary supports include microscope slides(e.g., glass microscope slides or plastic microscope slides), coverslips(e.g., glass coverslips or plastic coverslips), tissue culture dishes,multi-well plates, membranes (e.g., nitrocellulose or polyvinylidenefluoride (PVDF)) or BIACORE™ chips.

Exemplary methods involve determining in a prostate tissue sample from asubject the expression level of ERG and PTEN. In some examples, theexpression level of additional nucleic acids or proteins are determined,for example one or more other prostate cancer related markers, such asgrowth arrest-specific 1 (GAS 1; OMIM 139185), wingless-type MMTVintegration site family member 5 (WNT5A; OMIM 164975), thymidine kinase1 (TK1; OMIM 188300), V-raf murine sarcoma viral oncogene homolog B1(BRAF; OMIM 164757), ETS translocation variant 4 (ETV4; OMIM 600711),tumor protein p63 (OMIM 603273), BCL-2 (OMIM 151430), Ki67 (OMIM176741), ERK5 (OMIM 602521), prostate specific antigen (PSA; OMIM176820), ETV1 (OMIM 600541), measures of nuclear morphology (includingnuclear size and shape variation characteristics) or combinationsthereof. In addition, the expression level of one or more control genescan also be determined.

In exemplary methods, expression of ERG is increased and expression ofPTEN is decreased as compared to a standard value or a control sample.In other methods, the expression of another gene (e.g., WNT5A, TK1 orPSA) is increased. In some such methods, the relative increasedexpression of ERG and the relative decreased expression of PTENindicates, for example, a higher likelihood of prostate cancerprogression in the subject (e.g., a higher likelihood that the prostatecancer will invade the capsule in the subject), an increased likelihoodthat the prostate cancer will biochemically recur after surgery (e.g.,prostatectomy), an increased likelihood that the prostate cancer willmetastasize after surgery (e.g., prostatectomy), and/or a higherlikelihood that surgical treatment (e.g., prostatectomy) will fail, andan increased need for a non-surgical or alternate treatment for theprostate cancer.

In some methods, the expression of one or more genes of interest (e.g.,ERG and PTEN) is measured relative to a standard value or a controlsample. A standard values can include, without limitation, the averageexpression of the one or more genes of interest in a normal prostate orendothelial cells (e.g., calculated in an analogous manner to theexpression value of the genes in the prostate cancer sample), theaverage expression of the one or more genes of interest in a prostatesample obtained from a patient or patient population in which it isknown that prostate cancer did not invade the capsule, the averageexpression of the one or more genes of interest in a prostate sampleobtained from a region adjacent to the prostate cancer (e.g., normalprostate tissue adjacent to the cancer tissue); or the averageexpression of the one or more genes of interest in a prostate sampleobtained from a patient or patient population in which it is known thatprostate cancer did invade the capsule. A control sample can include,for example, normal prostate tissue or cells, prostate tissue or cellscollected from a patient or patient population in which it is known thatprostate cancer did not invade the capsule, prostate tissue or cellscollected from a patient or patient population in which it is known thatprostate cancer did invade the capsule, normal endothelial cells and/orendothelial cells collected from a patient or patient population inwhich it is known that prostate cancer did or did not invade thecapsule.

In other methods, expression of the gene(s) of interest is (are)measured in test (i.e., prostate cancer patient sample) and controlsamples relative to a value obtained for a control gene (e.g., one ormore of GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA(succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyltransferase 1), HBS1L (HBS1-like protein), β-actin, and AHSP (alphahaemoglobin stabilizing protein)) in each sample to produce normalizedtest and control values; then, the normalized value of the test sampleis compared to the normalized value of the control sample to obtain therelative expression of the gene(s) of interest (e.g., increasedexpression of ERG and decreased expression of PTEN). In some examples,expression of ERG and/or PTEN is normalized to expression of one or moreinternal controls, such as ERG expression in endothelial cells or PTENexpression in a normal gland.

An increase or decrease in gene expression may mean, for example, thatthe expression of a particular gene expression product (e.g., transcript(e.g., mRNA) or protein) in the test sample is at least about 20%, atleast about 25%, at least about 30%, at least about 50%, at least about75%, at least about 100%, at least about 150%, or at least about 200%higher or lower, respectively, of the applicable control (e.g., standardvalue or control sample). Alternatively, relative expression (i.e.,increase or decrease) may be in terms of fold difference; for example,the expression of a particular gene expression product (e.g., transcript(e.g., mRNA) or protein) in the test sample may be at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 8-fold, at least about 10-fold, at least about20-fold, at least about 50-fold, at least about 100-fold, or at leastabout 200-fold times higher or lower, respectively, of the applicablecontrol (e.g., standard value or control sample).

In some method embodiments where protein expression as determined byimmunohistochemistry is used as a measure of expression, scoring ofprotein expression may be semi-quantitative; for example, with proteinexpression levels recorded as 0, 1, 2, or 3 (including, in someinstances plus (or minus) values at each level, e.g., 1+, 2+, 3+) with 0being substantially no detectable protein expression and 3 (or 3+) beingthe highest detected protein expression. In such methods, an increase ordecrease in the expression is measured as a difference in the score ascompared the applicable control (e.g., standard value or controlsample); that is, a score of 3+ in a test sample as compared to a scoreof 0 for the control represents increased expression in the test sample,and a score of 0 in a test sample as compared to a score of 3+ for thecontrol represents decreased expression in the test sample.

Biochemical recurrence means the patient's PSA value has increasedprostate after prostatectomy or a PSA rise after a period of PSAdetection absence (for example after an initial (or subsequent)treatment(s), such as radiation treatment, chemotherapy, anti-hormonetreatment and/or surgery). Typically after an initial prostate cancertreatment PSA levels in the blood decrease to a stable and low leveland, in some instances, eventually become almost undetectable. In someexamples, recurrence of the prostate cancer is marked by rising PSAlevels (e.g., at least 0.2 ng/ml, at least 0.4 ng/ml, at least 0.6ng/ml, at least 1 ng/ml, or at least 2 ng/ml, or at least 5 ng/ml)and/or by identification of prostate cancer cells in the blood, prostatebiopsy or aspirate, in lymph nodes (e.g., in the pelvis or elsewhere) orat a metastatic site (e.g., muscles that help control urination, therectum, the wall of the pelvis, in bones or other organs).

Other exemplary methods predict the likelihood of prostate progression.Prostate cancer progression means that one or more indices of prostatecancer (e.g., serum PSA levels) show that the disease is advancingindependent of treatment. In some examples, prostate cancer progressionis marked by rising PSA levels (e.g., greater than 0.2 ng/mL) and/or byidentification of (or increasing numbers of) prostate cancer cells inthe blood, prostate biopsy or aspirate, in lymph nodes (e.g., in thepelvis or elsewhere) or at a metastatic site (e.g., muscles that helpcontrol urination, the rectum, the wall of the pelvis, in bones or otherorgans).

An increased likelihood of prostate cancer progression or prostatecancer recurrence can be quantified by any known metric. For example, anincreased likelihood can mean at least a 10% chance of occurring (suchas at least a 25% chance, at least a 50% chance, at least a 60% chance,at least a 75% chance or even greater than an 80% chance of occurring).

Some method embodiments are useful for prostate cancer prognosis, suchas prostate cancers that have penetrated the capsule. Prognosis is thelikely outcome of the disease (typically independent of treatment). ThePTEN expression (in some examples in combination with ERG expression)can be used to predict prostate cancer biochemical recurrence in asample collected well prior to such recurrence (such as a prostate CPsample). In some method embodiments, an increased likelihood ofbiochemical recurrence an increased likelihood of biochemical recurrencewithin 1 year, within 2 years, within 3 year, within 4 years, or within5 years after prostatectomy (for example as compared to a patient with aprostate cancer that is ERG+/PTEN+ or ERG−/PTEN+). Hence, such genesignature is a surrogate for the aggressiveness of the cancer withrecurring cancers being more aggressive. A poor (or poorer) prognosis islikely for a subject with a more aggressive cancer (such as having anincreased likelihood of metastasis or biochemical recurrence).

Still other method embodiments predict treatment outcome in prostatecancer patients, and are useful for directing (e.g., selecting useful)treatment modalities for prostate cancer patients, such as prostatecancers that have penetrated the capsule. As discussed elsewhere in thisspecification, expression of ERG and PTEN can be used to predict thatprostate cancer treatment (e.g., prostatectomy) is likely to fail (e.g.,the disease will recur). Hence, the disclosed gene signature(s) can beused by caregivers to counsel prostate cancer patients as to the likelysuccess of treatment (e.g., prostatectomy). Taken in the context of theparticular subject's medical history, the patient and the caregiver canmake better informed decisions of whether or not to treat (e.g., performsurgery, such as prostatectomy) and/or whether or not to providealternate treatment (such as, external beam radiotherapy, brachytherapy,chemotherapy, or watchful waiting).

1. Determining Expression Level (e.g., Gene Expression Profiling)

Expression levels may be determined or detected using any techniqueknown in the art. Exemplary techniques include, for example, methodsbased on hybridization analysis of polynucleotides (e.g., genomicnucleic acid sequences and/or transcripts (e.g., mRNA)), methods basedon sequencing of polynucleotides, methods based on detecting proteins(e.g., immunohistochemistry and proteomics-based methods). In someexamples, expression is measured or determined using a suitablyprogrammed computer or instrumentation (for example using the BenchMark(e.g., BenchMark ULTRA) and/or iScan Coreo Au slide scanner fromVentana). In some examples, expression of PTEN and ERG in the testsample is compared to a control using a suitably programmed computer(for example, PTEN and ERG expression values determined the test samplecan be compared to a control value of PTEN and ERG expression expectedin a normal prostate sample using a computer).

As discussed previously, gene expression levels may be affected byalterations in the genome (e.g., gene amplification, gene deletion, genefusion, or other chromosomal rearrangements or chromosome duplications(e.g., polysomy) or loss of one or more chromosomes). Accordingly, insome embodiments, gene expression levels may be inferred or determinedby detecting such genomic alterations. Genomic sequences harboring genesof interest may be quantified, for example, by in situ hybridization ofgene-specific genomic probes to chromosomes in a metaphase spread or aspresent in a cell nucleus. The making of gene-specific genomic probes iswell known in the art (see, e.g., U.S. Pat. Nos. 5,447,841, 5,756,696,6,872,817, 6,596,479, 6,500,612, 6,607,877, 6,344,315, 6,475,720,6,132,961, 7,115,709, 6,280,929, 5,491,224, 5,663,319, 5,776,688,5,663,319, 5,776,688, 6,277,569, 6,569,626, U.S. patent application Ser.No. 11/849,060, and PCT Appl. No. PCT/US07/77444). In some exemplarymethods, quantification of gene amplifications or deletions may befacilitated by comparing the number of binding sites for a gene-specificgenomic probe to a control genomic probe (e.g., a genomic probe specificfor the centromere of the chromosome upon which the gene of interest islocated). In some examples, gene amplification or deletion may bedetermined by the ratio of the gene-specific genomic probe to a control(e.g., centromeric) probe. For example, a ratio greater than two (suchas greater than three, greater than four, greater than five or ten orgreater) indicates amplification of the gene (or the chromosomal region)to which the gene-specific probe binds. In another example, a ratio lessthan one indicates deletion of the gene (or the chromosomal region) towhich the gene-specific probe binds. In particular method embodiments,it can be advantageous to also determine that gene amplification (orfusion) or deletion is accompanied by a corresponding increase ordecrease, respectively, in the expression products of the gene (e.g.,mRNA or protein); however, once a correlation is established, continuedco-detection is not needed (and may consume unnecessary resources andtime).

Gene expression levels also can be determined by quantification of genetranscript (e.g., mRNA). Commonly used methods known in the art for thequantification of mRNA expression in a sample include, withoutlimitation, northern blotting and in situ hybridization (e.g., Parkerand Barnes, Meth. Mol. Biol., 106:247-283, 1999)); RNAse protectionassays (e.g., Hod, Biotechniques, 13:852-854, 1992); and PCR-basedmethods, such as reverse transcription polymerase chain reaction(RT-PCR) (Weis et al., Trends in Genetics, 8:263-264, 1992) and realtime quantitative PCR, also referred to as qRT-PCR). Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes, orDNA-protein duplexes. Representative methods for sequencing-based geneexpression analysis include Serial Analysis of Gene Expression (SAGE),and gene expression analysis by massively parallel signature sequencing(MPSS).

Some method embodiments involving the determination of mRNA levelsutilize RNA (e.g., total RNA) isolated from a target sample, such aprostate cancer tissue sample. General methods for RNA (e.g., total RNA)isolation are well known in the art and are disclosed in standardtextbooks of molecular biology, including Ausubel et al., CurrentProtocols of Molecular Biology, John Wiley and Sons (1997). Methods forRNA extraction from paraffin-embedded tissues are disclosed in Examplesherein and, for example, by Rupp and Locker (Lab. Invest., 56:A67, 1987)and DeAndres et al. (BioTechniques, 18:42044, 1995). In particularexamples, RNA isolation can be performed using a purification kit,buffer set and protease obtained from commercial manufacturers, such asQiagen, according to the manufacturer's instructions. Other commerciallyavailable RNA isolation kits include MASTERPURE™ Complete DNA and RNAPurification Kit (EPICENTRE™ Biotechnologies) and Paraffin Block RNAIsolation Kit (Ambion, Inc.).

In the MassARRAY™ gene expression profiling method (Sequenom, Inc.),cDNA obtained from reverse transcription of total RNA is spiked with asynthetic DNA molecule (competitor), which matches the targeted cDNAregion in all positions, except a single base, and serves as an internalstandard. The cDNA/competitor mixture is amplified by standard PCR andis subjected to a post-PCR shrimp alkaline phosphatase (SAP) enzymetreatment, which results in the dephosphorylation of the remainingnucleotides. After inactivation of the alkaline phosphatase, the PCRproducts from the competitor and cDNA are subjected to primer extension,which generates distinct mass signals for the competitor- andcDNA-derived PCR products. After purification, these products aredispensed on a chip array, which is pre-loaded with components neededfor analysis with matrix-assisted laser desorption ionizationtime-of-flight (MALDI-TOF) mass spectrometry analysis. The cDNA presentin the reaction is then quantified by analyzing the ratios of the peakareas in the mass spectrum generated. For further details see, e.g.,Ding and Cantor, Proc. Natl. Acad. Sci. USA, 100:3059-3064, 2003.

Other methods for determining mRNA expression that involve PCR include,for example, differential display (Liang and Pardee, Science,257:967-971, 1992)); amplified fragment length polymorphism (Kawamoto etal., Genome Res., 12:1305-1312, 1999); BEADARRAY™ technology (Illumina,San Diego, Calif., USA; Oliphant et al., Discovery of Markers forDisease (Supplement to Biotechniques), June 2002; Ferguson et al., Anal.Chem., 72:5618, 2000; and Examples herein); XMAP™ technology (LuminexCorp., Austin, Tex., USA); BADGE assay (Yang et al., Genome Res.,11:1888-1898, 2001)); and high-coverage expression profiling (HiCEP)analysis (Fukumura et al., Nucl. Acids. Res., 31(16):e94, 2003).

Differential gene expression also can be determined using microarraytechniques. In these methods, specific binding partners, such as probes(including cDNAs or oligonucleotides) specific for RNAs of interest orantibodies specific for proteins of interest are plated, or arrayed, ona microchip substrate. The microarray is contacted with a samplecontaining one or more targets (e.g., mRNA or protein) for one or moreof the specific binding partners on the microarray. The arrayed specificbinding partners form specific detectable interactions (e.g., hybridizedor specifically bind to) their cognate targets in the sample ofinterest.

Serial analysis of gene expression (SAGE) is a method that allows thesimultaneous and quantitative analysis of a large number of genetranscripts, without the need of providing an individual hybridizationprobe for each transcript. In the SAGE method, a short sequence tag(about 10-14 bp) is generated that contains sufficient information touniquely identify a transcript, provided that the tag is obtained from aunique position within each transcript. Then, many transcripts arelinked together to form long serial molecules, that can be sequenced,revealing the identity of the multiple tags simultaneously. Theexpression pattern of any population of transcripts can be quantified bydetermining the abundance of individual tags, and identifying the genecorresponding to each tag (see, e.g., Velculescu et al., Science,270:484-487, 1995, and Velculescu et al., Cell, 88:243-51, 1997).

Gene expression analysis by massively parallel signature sequencing(MPSS) was first described by Brenner et al. (Nature Biotechnology,18:630-634, 2000). It is a sequencing approach that combinesnon-gel-based signature sequencing with in vitro cloning of millions oftemplates on separate 5 μm diameter microbeads. A microbead library ofDNA templates is constructed by in vitro cloning. This is followed bythe assembly of a planar array of the template-containing microbeads ina flow cell at a high density. The free ends of the cloned templates oneach microbead are analyzed simultaneously using a fluorescence-basedsignature sequencing method that does not require DNA fragmentseparation.

In some examples, differential gene expression is determined using insitu hybridization techniques, such as fluorescence in situhybridization (FISH) or chromogen in situ hybridization (CISH). In thesemethods, specific binding partners, such as probes labeled with aflouorphore or chromogen specific for a target gene, cDNA or mRNA (e.g.,a ERG or PTEN gene) is contacted with a sample, such as a prostatecancer sample mounted on a substrate (e.g., glass slide). The specificbinding partners form specific detectable interactions (e.g., hybridizedto) their cognate targets in the sample. For example, hybridizationbetween the probes and the target nucleic acid can be detected, forexample by detecting a label associated with the probe. In someexamples, microscopy, such as fluorescence microscopy, is used.

Immunohistochemistry (IHC) is one exemplary technique useful fordetecting protein expression products in the disclosed methods.Antibodies (e.g., monoclonal and/or polyclonal antibodies) specific foreach protein are used to detect expression. The antibodies can bedetected by direct labeling of the antibodies themselves, for example,with radioactive labels, fluorescent labels, hapten labels such as,biotin, or an enzyme such as horseradish peroxidase or alkalinephosphatase. Alternatively, unlabeled primary antibody is used inconjunction with a labeled secondary antibody, comprising antisera,polyclonal antisera or a monoclonal antibody specific for the primaryantibody. IHC protocols and kits are well known in the art and arecommercially available.

Proteomic analysis is another exemplary technique useful for detectingprotein expression products in the disclosed methods. The term“proteome” is defined as the totality of the proteins present in asample (e.g., tissue, organism, or cell culture) at a certain point oftime. Proteomics includes, among other things, study of the globalchanges of protein expression in a sample (also referred to as“expression proteomics”). An exemplary proteomics assay involves (i)separation of individual proteins in a sample, e.g., by 2-D gelelectrophoresis; (ii) identification of the individual proteinsrecovered from the gel, e.g., by mass spectrometry or N-terminalsequencing, and (iii) analysis of the data.

2. Outputting Expression Level

Following the measurement of the expression levels of one or more of themolecules described herein, the assay results, findings, diagnoses,predictions and/or treatment recommendations are typically recorded andcommunicated to technicians, physicians and/or patients, for example. Incertain embodiments, computers will be used to communicate suchinformation to interested parties, such as, patients and/or theattending physicians. Based on the measurement, the therapy administeredto a subject can be modified.

In one embodiment, a diagnosis, prediction and/or treatmentrecommendation based on the expression level in a test subject of ERG orPTEN (or in combination with other diagnostics, such as those on aprostate cancer nomogram, such as PSA value and Gleason grade) iscommunicated to interested parties as soon as possible after the assayis completed and the diagnosis and/or prediction is generated. Theresults and/or related information may be communicated to the subject bythe subject's treating physician. Alternatively, the results may becommunicated directly to interested parties by any means ofcommunication, including writing, such as by providing a written report,electronic forms of communication, such as email, or telephone.Communication may be facilitated by use of a suitably programmedcomputer, such as in case of email communications. In certainembodiments, the communication containing results of a diagnostic testand/or conclusions drawn from and/or treatment recommendations based onthe test, may be generated and delivered automatically to interestedparties using a combination of computer hardware and software which willbe familiar to artisans skilled in telecommunications. One example of ahealthcare-oriented communications system is described in U.S. Pat. No.6,283,761; however, the present disclosure is not limited to methodswhich utilize this particular communications system. In certainembodiments of the methods of the disclosure, all or some of the methodsteps, including the assaying of samples, diagnosing or prognosis ofprostate cancer, and communicating of assay results or diagnoses orprognosis, may be carried out in diverse (e.g., foreign) jurisdictions.

B. Capsular Penetration Biomarkers

1. Ets Related Gene (ERG) (OMIM: 165080)

The human Ets related gene (ERG) (also known as erg-3 and p55) islocated on chromosome 21 (21q22.3) and is a member of the ETS family oftranscription factors. ERG sequences are publically available, forexample from GenBank® (e.g., accession numbers NP_001129626 andNP_598420.1 (proteins) and NM_133659.2, NM_001136154.1, andNM_001838708.2 (nucleic acids)).

ERG protein (see, e.g., SEQ ID NO: 2) is a transcriptional regulatorthat binds purine-rich sequences. Fusion of the ERG gene with othergenes has been shown to be associated with different cancers. Forexample, the t(16;21)(p11;q22) translocation of the ERG gene fused withthe fused in sarcoma (FUS) gene is associated with human myeloidleukemia. EWS-ERG fusions are associated with the Ewing family oftumors. ERG fusion with the 5′ untranslated region of transmembraneprotease, serine 2 (TMPRSS2) (located on human chromosome 21) areassociated with prostate cancer. The TMPRS22 and ERG genes are arrangedtandemly on chromosome 21q22. The TMPRSS2/ERG fusion joins TMPRSS2 exons1 or 2 usually to ERG exons 2, 3 or 4, which results in activation ofthe ERG transcription factor. TMPRSS/ERG rearrangements occur in about50% of prostate cancers and 20% of high-grade prostatic intraepithelialneoplasia (HGPIN) lesions, resulting in upregulation of ERG. TMPRSS/ERGrearrangement results in a PIN like lesion which can be converted to aninvasive state by up-regulation of the PI3K pathway.

2. Phosphatase and Tensin Homolog (PTEN) (OMIM 601728)

The human PTEN gene is located on chromosome 10 and the mouse PTEN geneis located on chromosome 19. PTEN sequences (both wild-type and mutant)are publically available, for example from GenBank® (e.g., accessionnumbers NP_000305.3, AAD13528.1, EAW50174.1, EAW50173.1, EAW50172.1,AAH05821.1 and NP_032986.1 (proteins) and NM_000314.4 and NM_008960.2(nucleic acids)).

PTEN, also referred to as MMAC (mutated in multiple advanced cancers)phosphatase, is a tumor suppressor gene implicated in a wide variety ofhuman cancers. The PTEN protein (e.g., SEQ ID NO: 4) is aphosphatidylinositol-3,4,5-trisphosphate 3-phosphatase, which includes atensin-like domain as well as a catalytic domain. Unlike most proteintyrosine phosphatases, PTEN preferentially dephosphorylatesphosphoinositide substrates. PTEN negatively regulates intracellularlevels of phosphatidylinositol-3, 4, 5-trisphosphate in cells andfunctions as a tumor suppressor by negatively regulating Akt/PKBsignaling pathway. Mutations and deletions of PTEN have been shown to beassociated with cancers.

3. Variant Sequences

In addition to the specific sequences provided herein (e.g., SEQ ID NOS:1-4), and the sequences which are currently publically available, oneskilled in the art will appreciate that variants of such sequences maybe present in a particular subject. For example, polymorphisms for aparticular gene or protein may be present. In addition, a sequence mayvary between different organisms. In particular examples, a variantsequence retains the biological activity of its corresponding nativesequence. For example, a ERG or PTEN sequence present in a particularsubject may can have conservative amino acid changes (such as, veryhighly conserved substitutions, highly conserved substitutions orconserved substitutions), such as 1 to 5 or 1 to 10 conservative aminoacid substitutions. Exemplary conservative amino acid substitutions areshown in Table 1.

TABLE 1 Exemplary conservative amino acid substitutions. HighlyConserved Conserved Very Highly - Substitutions Substitutions OriginalConserved (from the (from the Residue Substitutions Blosum90 Matrix)Blosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg LysGln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Lys,Arg, Asp, Gln, Glu, His, Ser, Thr Lys, Ser, Thr Asp Glu Asn, Glu Asn,Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Arg, Asn,Asp, Glu, His, Lys, Met Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn,Asp, Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln,Tyr Arg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met, Phe,Val Leu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys Arg; Gln; GluArg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln, Ile, Leu,Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, Tyr Ile, Leu,Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu, Gly, Lys,Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, Tyr Phe, TyrTyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala,Ile, Leu, Met, Thr

In some embodiments, an ERG or PTEN sequence is a sequence variant of anative ERG or PTEN sequence, respectively, such as a nucleic acid orprotein sequence that has at least 99%, at least 98%, at least 95%, atleast 92%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, or at least 60% sequence identity to thesequences set forth in SEQ ID NOS: 1-4 (or such amount of sequenceidentity to a GenBank® accession number referred to herein) wherein theresulting variant retains ERG or PTEN biological activity. “Sequenceidentity” is a phrase commonly used to describe the similarity betweentwo amino acid sequences (or between two nucleic acid sequences).Sequence identity typically is expressed in terms of percentageidentity; the higher the percentage, the more similar the two sequences.

Methods for aligning sequences for comparison and determining sequenceidentity are well known in the art. Various programs and alignmentalgorithms are described in: Smith and Waterman, Adv. Appl. Math.,2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearsonand Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins andSharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153,1989; Corpet et al., Nucleic Acids Research, 16:10881-10890, 1988;Huang, et al., Computer Applications in the Biosciences, 8:155-165,1992; Pearson et al., Methods in Molecular Biology, 24:307-331, 1994;Tatiana et al., FEMS Microbiol. Lett., 174:247-250, 1999. Altschul etal. present a detailed consideration of sequence-alignment methods andhomology calculations (J. Mol. Biol., 215:403-410, 1990).

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™, Altschul et al., J. Mol. Biol.,215:403-410, 1990) is publicly available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the Internet, for use in connection with the sequence-analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe internet under the help section for BLAST™.

For comparisons of amino acid sequences of greater than about 15 aminoacids, the “Blast 2 sequences” function of the BLAST™ (Blastp) programis employed using the default BLOSUM62 matrix set to default parameters(cost to open a gap [default=5]; cost to extend a gap [default=2];penalty for a mismatch [default=3]; reward for a match [default=1];expectation value (E) [default=10.0]; word size [default=3]; and numberof one-line descriptions (V) [default=100]. When aligning short peptides(fewer than around 15 amino acids), the alignment should be performedusing the Blast 2 sequences function “Search for short nearly exactmatches” employing the PAM30 matrix set to default parameters (expectthreshold=20000, word size=2, gap costs: existence=9 and extension=1)using composition-based statistics.

C. Use in Combination with Other Diagnostic and Prognostic Assays

The disclosed methods can be used in combination with one or more otherassays that are used to diagnose or prognose prostate cancer outcomes.For example, a prostate cancer patient's Gleason scores based on ahistopathological review, PSA scores, and nomograms (such as the PartinCoefficient Tables) can be used in combination with the disclosedmethods to allow for enhanced diagnostic and prognostic capabilities ofprostate cancer, such as those that have penetrated the capsule. Thus,in some examples, the disclosed methods further include measuring thepatient's PSA level, determining the patient's nomogram, or both.

For example, prostate cancer nomograms can be used to predict theprobability that a patient's cancer will recur (for example afterradical prostatectomy), that is, the probability at one, two, five,seven and 10 years that the patient's serum PSA level will becomedetectable and begin to rise steadily. Nomograms include information onone or more of the patient's pre-treatment PSA, age, Gleason grade(primary, secondary and sum), year of prostatectomy, months free ofcancer, whether or not the surgical margins were positive, whether ornot there was extra capsular extension (penetration); whether or notthere was seminal vesicle involvement, whether or not there was lymphnode involvement, whether or not the patient receive neoadjuvanthormones, and whether whether or not the patient receive radiationtherapy before the radical prostatectomy. Thus, the patient's ERG andPTEN expression levels can be incorporated into currently availablenomograms to further increase the accuracy of such predictions.

D. Follow-Up Therapies

The disclosed methods can further include selecting subjects fortreatment for prostate cancer, for example if the sample is diagnosed ashaving an aggressive prostate cancer (EGR+/PTEN−). Alternatively, thedisclosed methods can further include selecting subjects for notreatment, if the sample is diagnosed as a non-aggressive prostatecancer (e.g., EGR−/PTEN+).

In some embodiments, the disclosed methods include one or more of thefollowing depending on the patient's diagnosis or prognosis: a)prescribing a treatment regimen for the subject if the subject'sdetermined diagnosis/prognosis is having an aggressive prostate cancer(such as treatment with one or more radiotherapies and/orchemotherapeutic agents, additional surgery, or combinations thereof);b) not prescribing a treatment regimen for the subject if the subject'sdetermined diagnosis/prognosis is a non-aggressive prostate cancer; c)administering a treatment (such as treatment with one or moreradiotherapies and/or chemotherapeutic agents, additional surgery, orcombinations thereof) to the subject if the subject's determineddiagnosis/prognosis is having an aggressive prostate cancer; and d) notadministering a treatment regimen to the subject if the subject'sdetermined diagnosis/prognosis is a non-aggressive prostate cancer. Inan alternative embodiment, the method can include recommending one ormore of (a)-(d). Thus, the disclosed methods can further includetreating a subject for prostate cancer.

D. Compositions

Disclosed herein are genes (ERG and PTEN) the expression of whichcharacterizes prostate cancer that has penetrated the capsule insubjects afflicted with the disease. Accordingly, compositions thatfacilitate the detection of such genes in biological samples are nowenabled.

1. Kits

Kits useful for facilitating the practice of a disclosed method are alsocontemplated. In one embodiment, a kit is provided for detecting PTENand ERG nucleic acid or protein molecules, for example in combinationwith one to ten (e.g., 1, 2, 3, 4, or 5) control genes or proteins(e.g., β-actin, GAPDH, SDHA, HPRT1, HBS1L, AHSP or combinationsthereof). In yet other specific examples, kits are provided fordetecting only PTEN and ERG nucleic acid or protein molecules. Thedetection means can include means for detecting a genomic alterationinvolving the gene and/or a gene expression product, such as an mRNA orprotein. In particular examples, means for detecting a least ERG andPTEN are packaged in separate containers or vials. In some examples,means for detecting one or more ERG and PTEN genes or proteins arepresent on an array (discussed below).

Exemplary kits can include at least one means for detection of ERG andPTEN genes or gene products (for example in combination with otherprostate cancer related genes/proteins or control genes/proteins) suchas, at least two, at least three, at least four, or at least fivedetection means), such as means that permit detection of at least ERGand PTEN. In some examples, such kits can further include at least onemeans for detection of one or more (e.g., one to three) control genes orproteins. Detection means can include, without limitation, a nucleicacid probe specific for a genomic sequence including a disclosed gene, anucleic acid probe specific for a transcript (e.g., mRNA) encoded by adisclosed gene, a pair of primers for specific amplification of adisclose gene (e.g., genomic sequence or cDNA sequence of such gene), anantibody or antibody fragment specific for a protein encoded by adisclosed gene.

In one example a kit can include means for detecting in a biologicalsample an ERG genomic sequence, ERG transcript or ERG protein, and meansfor detecting in a biological sample a PTEN genomic sequence, PTENtranscript or PTEN protein, or any combination thereof. For example, thekit can include a means for detecting in a biological sample an ERGtranscript or protein and a means for detecting in a biological sample aPTEN transcript or protein, such as a nucleic acid probe specific for anERG transcript and a nucleic acid probe specific for a PTEN transcript,such as a pair of primers for specific amplification of an ERGtranscript and a pair of primers for specific amplification of a PTENtranscript, or such as an antibody specific for ERG protein and anantibody specific for a PTEN protein.

Particular kit embodiments can include, for instance, one or more (suchas two, three, or four) detection means selected from a nucleic acidprobe specific for an ERG transcript, a nucleic acid probe specific fora PTEN transcript, a pair of primers for specific amplification of anERG transcript, a pair of primers for specific amplification of a PTENtranscript, an antibody specific for an ERG protein, and an antibodyspecific for a PTEN protein. Particular kit embodiments can furtherinclude, for instance, one or more (such as two or three) detectionmeans selected from a nucleic acid probe specific for a controltranscript, a pair of primers for specific amplification of controltranscript, and an antibody specific for control protein. Exemplarycontrol genes/proteins include GAPDH, SDHA, HPRT1, HBS1L, β-actin, andAHSP. In some examples, kits can further include, for instance, one ormore (such as two or three) detection means selected from a nucleic acidprobe specific for a prostate cancer related transcript, a pair ofprimers for specific amplification of prostate cancer relatedtranscript, and an antibody specific for prostate cancer relatedprotein. Exemplary prostate cancer related genes/proteins include GAS 1;WNT5A; TK1; BRAF; ETV4; tumor protein p63; BCL-2; Ki67; ERK5; and PSA.

In some kit embodiments, the primary detection means (e.g., nucleic acidprobe, nucleic acid primer, or antibody) can be directly labeled, e.g.,with a fluorophore, chromophore, or enzyme capable of producing adetectable product (such as alkaline phosphates, horseradish peroxidaseand others commonly know in the art).

Other kit embodiments will include secondary detection means; such assecondary antibodies (e.g., goat anti-rabbit antibodies, rabbitanti-mouse antibodies, anti-hapten antibodies) or non-antibodyhapten-binding molecules (e.g., avidin or streptavidin). In some suchinstances, the secondary detection means will be directly labeled with adetectable moiety. In other instances, the secondary (or higher order)antibody will be conjugated to a hapten (such as biotin, DNP, and/orFITC), which is detectable by a detectably labeled cognate haptenbinding molecule (e.g., streptavidin (SA) horseradish peroxidase, SAalkaline phosphatase, and/or SA QDot Nanocrystals™). Some kitembodiments may include colorimetric reagents (e.g., DAB, and/or AEC) insuitable containers to be used in concert with primary or secondary (orhigher order) detection means (e.g., antibodies) that are labeled withenzymes for the development of such colorimetric reagents.

In some embodiments, a kit includes positive or negative controlsamples, such as a cell line or tissue known to express or not expressPTEN or ERG. In particular examples, control samples are FFPE. Exemplarysamples include but are not limited to normal (e.g., non cancerous)cells or tissues, breast cancer cell lines or tissues, prostate cancersamples from subject known not to have capsular penetration, andprostate cancer samples from subject known to have capsular penetration.

In some embodiments, a kit includes instructional materials disclosing,for example, means of use of a nucleic acid probe or antibody thatspecifically binds a PTEN or ERG (e.g., mRNA or protein), or means ofuse for a particular primer or probe. The instructional materials may bewritten, in an electronic form (e.g., computer diskette or compact disk)or may be visual (e.g., video files). The kits may also includeadditional components to facilitate the particular application for whichthe kit is designed. Thus, for example, the kit can include buffers andother reagents routinely used for the practice of a particular disclosedmethod. Such kits and appropriate contents are well known to those ofskill in the art.

Certain kit embodiments can include a carrier means, such as a box, abag, a satchel, plastic carton (such as molded plastic or other clearpackaging), wrapper (such as, a sealed or sealable plastic, paper, ormetallic wrapper), or other container. In some examples, kit componentswill be enclosed in a single packaging unit, such as a box or othercontainer, which packaging unit may have compartments into which one ormore components of the kit can be placed. In other examples, a kitincludes a one or more containers, for instance vials, tubes, and thelike that can retain, for example, one or more biological samples to betested.

Other kit embodiments include, for instance, syringes, cotton swabs, orlatex gloves, which may be useful for handling, collecting and/orprocessing a biological sample. Kits may also optionally containimplements useful for moving a biological sample from one location toanother, including, for example, droppers, syringes, and the like. Stillother kit embodiments may include disposal means for discarding used orno longer needed items (such as subject samples, etc.). Such disposalmeans can include, without limitation, containers that are capable ofcontaining leakage from discarded materials, such as plastic, metal orother impermeable bags, boxes or containers.

2. Arrays

Microarrays for the detection of genes (e.g., genomic sequence andcorresponding transcripts) and proteins are well known in the art.Microarrays include a solid surface (e.g., glass slide) upon which many(e.g., hundreds or even thousands) of specific binding agents (e.g.,cDNA probes, mRNA probes, or antibodies) are immobilized. The specificbinding agents are distinctly located in an addressable (e.g., grid)format on the array. The number of addressable locations on the arraycan vary, for example from at least two, to at least 10, at least 20, atleast 30, at least 33, at least 40, at least 50, at least 75, at least100, at least 150, at least 200, at least 300, at least 500, least 550,at least 600, at least 800, at least 1000, at least 10,000, or more. Thearray is contacted with a biological sample believed to contain targets(e.g., mRNA, cDNA, or protein, as applicable) for the arrayed specificbinding agents. The specific binding agents interact with their cognatetargets present in the sample. The pattern of binding of targets amongall immobilized agents provides a profile of gene expression. Inparticular embodiments, various scanners and software programs can beused to profile the patterns of genes that are “turned on” (e.g., boundto an immobilized specific binding agent). Representative microarraysare described, e.g., in U.S. Pat. Nos. 5,412,087, 5,445,934, 5,744,305,6,897,073, 7,247,469, 7,166,431, 7,060,431, 7,033,754, 6,998,274,6,942,968, 6,890,764, 6,858,394, 6,770,441, 6,620,584, 6,544,732,6,429,027, 6,396,995, and 6,355,431.

Disclosed herein are arrays, whether protein or nucleic acid arrays, forthe detection at least ERG and PTEN. Particular array embodimentsconsist of nucleic probes or antibodies specific for ERG and PTENexpression products and one or more control products (e.g., mRNA, cDNAor protein). More particular array embodiments consist of nucleic probesor antibodies specific for ERG and PTEN expression products (e.g., mRNA,cDNA or protein) and one or more prostate cancer related products (e.g.,mRNA, cDNA or protein). More particular array embodiments consist ofnucleic probes or antibodies specific for ERG and PTEN expressionproducts, one or more control products, and one or more prostate cancerrelated products (e.g., mRNA, cDNA or protein). More particular arrayembodiments consist of nucleic probes or antibodies specific for ERG andPTEN expression products (e.g., mRNA, cDNA or protein).

a. Nucleic Acid Arrays

In one example, the array includes nucleic acid probes that canhybridize to at least ERG and PTEN nucleic acids (such as genes). Inparticular examples, an array further includes probes that canspecifically hybridize to other prostate marker nucleic acids, such asone or more of GAS 1; WNT5A; TK1; BRAF; ETV4; tumor protein p63; BCL-2;Ki67; ERK5; and PSA. Certain of such arrays (as well as the methodsdescribed herein) can further include oligonucleotides specific forcontrol genes (e.g., one or more of GAPDH, SDHA, HPRT1, HBS1L, β-actin,and AHSP).

In one example, a set of oligonucleotide probes is attached to thesurface of a solid support for use in detection of ERG and PTEN, such asdetection of nucleic acid sequences (such as genomic DNA, cDNA or mRNA)obtained from the subject (e.g., from a prostate cancer sample).Additionally, if an internal control nucleic acid sequence is used (suchas a nucleic acid sequence obtained from a subject who has not hadprostate cancer or a control gene nucleic acid sequence) a nucleic acidprobe can be included to detect the presence of this control nucleicacid molecule.

The oligonucleotide probes bound to the array can specifically bindsequences obtained from the subject (such as a sample containing nucleicacids or nucleic acids isolated from the sample), or amplified from thesubject, such as under high stringency conditions. Agents of use withthe method include oligonucleotide probes that recognize ERG (includingfusions of ERG, such as TMPRSS/ERG rearrangements). Such sequences canbe determined by examining the known gene sequences, and choosing probesequences that specifically hybridize to ERG or TMPRSS/ERGrearrangements, but not other gene sequences.

The methods and apparatus in accordance with the present disclosure takeadvantage of the fact that under appropriate conditions oligonucleotideprobes form base-paired duplexes with nucleic acid molecules that have acomplementary base sequence. The stability of the duplex is dependent ona number of factors, including the length of the oligonucleotide probe,the base composition, and the composition of the solution in whichhybridization is effected. The effects of base composition on duplexstability can be reduced by carrying out the hybridization in particularsolutions, for example in the presence of high concentrations oftertiary or quaternary amines. The thermal stability of the duplex isalso dependent on the degree of sequence similarity between thesequences. By carrying out the hybridization at temperatures close tothe anticipated T_(m)'s of the type of duplexes expected to be formedbetween the target sequences and the oligonucleotides bound to thearray, the rate of formation of mis-matched duplexes may besubstantially reduced.

The length of each oligonucleotide probe employed in the array can beselected to optimize binding of target sequences. An optimum length foruse with a particular gene sequence under specific screening conditionscan be determined empirically. Thus, the length for each individualelement of the set of oligonucleotide sequences included in the arraycan be optimized for screening. In one example, oligonucleotide probesare at least 12 nucleotides (nt) in length, for example at least 20 nt,at least 50 nt, at least 100 nt, at least 1000 nt, at least 10,000 nt,or at least 100,000 nt, such as from about 20 to about 35 nt in length,about 25 to about 40 nt in length, about 25 to about 100 nt in length,about 1000 to about 6000 nt in length, about 10,000 to about 50,000 ntin length, about 10,000 to about 100,000 in length, about 10,000 toabout 500,000 in length, or about 10,000 to about 1,000,000 in length.

The oligonucleotide probe sequences forming the array can be directlylinked to the support. Alternatively, the oligonucleotide probes can beattached to the support by oligonucleotides (that do notnon-specifically hybridize to the target gene sequences) or othermolecules that serve as spacers or linkers to the solid support.

b. Protein Arrays

In another example, an array includes protein sequences (or a fragmentof such proteins, or antibodies specific to such proteins or proteinfragments), which specifically bind to ERG and PTEN, for example proteinbinding agents that can specifically bind to ERG and PTEN. In particularexamples, an array includes protein binding agents that can recognizeadditional prostate cancer biomarkers, such as one or more of GAS 1;WNT5A; TK1; BRAF; ETV4; ETV1; tumor protein p63; BCL-2; Ki67; ERK5; andPSA. Certain of such arrays (as well as the methods described herein)can further include protein binding agents specific for control proteins(e.g., one or more of GAPDH, SDHA, HPRT1, HBS1L, β-actin, and AHSP).

The proteins or antibodies forming the array can be directly linked tothe support. Alternatively, the proteins or antibodies can be attachedto the support by spacers or linkers to the solid support. Changes inprotein expression can be detected using, for instance, aprotein-specific binding agent, which in some instances is labeled. Incertain examples, detecting a change in protein expression includescontacting a protein sample obtained from a prostate cancer sample of asubject with a protein-specific binding agent (which can be for examplepresent on an array); and detecting whether the binding agent is boundby the sample and thereby measuring the levels of the target proteinpresent in the sample. A increase in the level of ERG and a decrease inthe level of PTEN in the test sample, relative to the level of the sameprotein found an analogous control sample (e.g., from a subject who doesnot have prostate cancer, or a non-cancerous prostate sample from thesame patient which is adjacent to the prostate cancer), in particularexamples indicates that the subject has a poor prognosis.

c. Array Substrate

The array solid support can be formed from an organic polymer. Suitablematerials for the solid support include, but are not limited to:polypropylene, polyethylene, polybutylene, polyisobutylene,polybutadiene, polyisoprene, polyvinylpyrrolidine,polytetrafluroethylene, polyvinylidene difluroide,polyfluoroethylene-propylene, polyethylenevinyl alcohol,polymethylpentene, polycholorotrifluoroethylene, polysulformes,hydroxylated biaxially oriented polypropylene, aminated biaxiallyoriented polypropylene, thiolated biaxially oriented polypropylene,etyleneacrylic acid, thylene methacrylic acid, and blends of copolymersthereof (e.g., U.S. Pat. No. 5,985,567).

In general, suitable characteristics of the material that can be used toform the solid support surface include: being amenable to surfaceactivation such that upon activation, the surface of the support iscapable of covalently attaching a biomolecule such as an oligonucleotideor antibody thereto; amenability to “in situ” synthesis of biomolecules;being chemically inert such that at the areas on the support notoccupied by the oligonucleotides or antibodies are not amenable tonon-specific binding, or when non-specific binding occurs, suchmaterials can be readily removed from the surface without removing theoligonucleotides or antibodies.

In one example, the solid support surface is polypropylene.Polypropylene is chemically inert and hydrophobic. Non-specific bindingis generally avoidable, and detection sensitivity is improved.Polypropylene has good chemical resistance to a variety of organic acids(such as formic acid), organic agents (such as acetone or ethanol),bases (such as sodium hydroxide), salts (such as sodium chloride),oxidizing agents (such as peracetic acid), and mineral acids (such ashydrochloric acid). Polypropylene also provides a low fluorescencebackground, which minimizes background interference and increases thesensitivity of the signal of interest.

In another example, a surface activated organic polymer is used as thesolid support surface. One example of a surface activated organicpolymer is a polypropylene material aminated via radio frequency plasmadischarge. Such materials are easily utilized for the attachment ofnucleic acid molecules. The amine groups on the activated organicpolymers are reactive with nucleotide molecules such that the nucleotidemolecules can be bound to the polymers. Other reactive groups can alsobe used, such as carboxylated, hydroxylated, thiolated, or active estergroups.

d. Array Formats

A wide variety of array formats can be employed in accordance with thepresent disclosure. One example includes a linear array ofoligonucleotide bands, generally referred to in the art as a dipstick.Another suitable format includes a two-dimensional pattern of discretecells (such as 4096 squares in a 64 by 64 array). As is appreciated bythose skilled in the art, other array formats including, but not limitedto slot (rectangular) and circular arrays are equally suitable for use(e.g., U.S. Pat. No. 5,981,185). In one example, the array is formed ona polymer medium, which is a thread, membrane or film. An example of anorganic polymer medium is a polypropylene sheet having a thickness onthe order of about 1 mil. (0.001 inch) to about 20 mil., although thethickness of the film is not critical and can be varied over a fairlybroad range. Particularly disclosed for preparation of arrays arebiaxially oriented polypropylene (BOPP) films; in addition to theirdurability, BOPP films exhibit a low background fluorescence.

The array formats of the present disclosure can be included in a varietyof different types of formats. A “format” includes any format to whichthe solid support can be affixed, such as microtiter plates, test tubes,inorganic sheets, dipsticks, and the like. For example, when the solidsupport is a polypropylene thread, one or more polypropylene threads canbe affixed to a plastic dipstick-type device; polypropylene membranescan be affixed to glass slides. The particular format is, in and ofitself, unimportant. All that is necessary is that the solid support canbe affixed thereto without affecting the functional behavior of thesolid support or any biopolymer absorbed thereon, and that the format(such as the dipstick or slide) is stable to any materials into whichthe device is introduced (such as clinical samples and hybridizationsolutions).

The arrays of the present disclosure can be prepared by a variety ofapproaches. In one example, oligonucleotide or protein sequences aresynthesized separately and then attached to a solid support (e.g., seeU.S. Pat. No. 6,013,789). In another example, sequences are synthesizeddirectly onto the support to provide the desired array (e.g., see U.S.Pat. No. 5,554,501). Suitable methods for covalently couplingoligonucleotides and proteins to a solid support and for directlysynthesizing the oligonucleotides or proteins onto the support are knownto those working in the field; a summary of suitable methods can befound in Matson et al., Anal. Biochem. 217:306-10, 1994. In one example,the oligonucleotides are synthesized onto the support using conventionalchemical techniques for preparing oligonucleotides on solid supports(e.g., see PCT applications WO 85/01051 and WO 89/10977, or U.S. Pat.No. 5,554,501).

A suitable array can be produced using automated means to synthesizeoligonucleotides in the cells of the array by laying down the precursorsfor the four bases in a predetermined pattern. Briefly, amultiple-channel automated chemical delivery system is employed tocreate oligonucleotide probe populations in parallel rows (correspondingin number to the number of channels in the delivery system) across thesubstrate. Following completion of oligonucleotide synthesis in a firstdirection, the substrate can then be rotated by 90° to permit synthesisto proceed within a second (2°) set of rows that are now perpendicularto the first set. This process creates a multiple-channel array whoseintersection generates a plurality of discrete cells.

Oligonucleotide probes can be bound to the support by either the 3′ endof the oligonucleotide or by the 5′ end of the oligonucleotide. In oneexample, the oligonucleotides are bound to the solid support by the 3′end. However, one of skill in the art can determine whether the use ofthe 3′ end or the 5′ end of the oligonucleotide is suitable for bondingto the solid support. In general, the internal complementarity of anoligonucleotide probe in the region of the 3′ end and the 5′ enddetermines binding to the support. In particular examples, theoligonucleotide probes on the array include one or more labels, thatpermit detection of oligonucleotide probe:target sequence hybridizationcomplexes.

3. Protein Specific Binding Agents

In some examples, the means used to detect ERG or PTEN is a proteinspecific binding agent, such as an antibody or fragment thereof. Forexample, antibodies or aptamers specific for ERG or PTEN (e.g., SEQ IDNO: 2, or 4, respectively), can be obtained from a commerciallyavailable source or prepared using techniques common in the art. Suchspecific binding agents can also be used in the prognostic methodsprovided herein.

Specific binding reagents include, for example, antibodies or functionalfragments or recombinant derivatives thereof, aptamers, mirror-imageaptamers, or engineered nonimmunoglobulin binding proteins based on anyone or more of the following scaffolds: fibronectin (e.g., ADNECTINS™ ormonobodies), CTLA-4 (e.g., EVIBODIES™), tendamistat (e.g., McConnell andHoess, J. Mol. Biol., 250:460-470, 1995), neocarzinostatin (e.g., Heydet al., Biochem., 42:5674-83, 2003), CBM4-2 (e.g., Cicortas-Gunnarssonet al., Protein Eng. Des. Sel., 17:213-21, 2004), lipocalins (e.g.,ANTICALINS™; Schlehuber and Skerra, Drug Discov. Today, 10:23-33, 2005),T-cell receptors (e.g., Chlewicki et al., J. Mol. Biol., 346:223-39,2005), protein A domain (e.g., AFFIBODIES™; Engfeldt et al.,ChemBioChem, 6:1043-1050, 2005), Im9 (e.g., Bernath et al., J. Mol.Biol., 345:1015-26, 2005), ankyrin repeat proteins (e.g., DARPins;Amstutz et al., J. Biol. Chem., 280:24715-22, 2005), tetratricopeptiderepeat proteins (e.g., Cortajarena et al., Protein Eng. Des. Sel.,17:399-409, 2004), zinc finger domains (e.g., Bianchi et al., J. Mol.Biol., 247:154-60, 1995), pVIII (e.g., Petrenko et al., Protein Eng.,15:943-50, 2002), GCN4 (Sia and Kim, Proc. Natl Acad. Sci. USA,100:9756-61, 2003), avian pancreatic polypeptide (APP) (e.g., Chin etal., Bioorg. Med. Chem. Lett., 11:1501-5, 2001), WW domains, (e.g.,Dalby et al., Protein Sci., 9:2366-76, 2000), SH3 domains (e.g.,Hiipakka et al., J. Mol. Biol., 293:1097-106, 1999), SH2 domains(Malabarba et al., Oncogene, 20:5186-5194, 2001), PDZ domains (e.g.,TELOBODIES™; Schneider et al., Nat. Biotechnol., 17:170-5, 1999), TEM-1β-lactamase (e.g., Legendre et al., Protein Sci., 11:1506-18, 2002),green fluorescent protein (GFP) (e.g., Zeytun et al., Nat. Biotechnol.,22:601, 2004), thioredoxin (e.g., peptide aptamers; Lu et al.,Biotechnol., 13:366-372, 1995), Staphylococcal nuclease (e.g., Norman,et al., Science, 285:591-5, 1999), PHD fingers (e.g., Kwan et al.,Structure, 11:803-13, 2003), chymotrypsin inhibitor 2 (CI2) (e.g.,Karlsson et al., Br. J. Cancer, 91:1488-94, 2004), bovine pancreatictrypsin inhibitor (BPTI) (e.g., Roberts, Proc. Natl. Acad. Sci. USA,89:2429-33, 1992) and many others (see review by Binz et al., Nat.Biotechnol., 23(10):1257-68, 2005 and supplemental materials).

Specific binding reagents also include antibodies. The term “antibody”refers to an immunoglobulin molecule (or combinations thereof) thatspecifically binds to, or is immunologically reactive with, a particularantigen, and includes polyclonal, monoclonal, genetically engineered andotherwise modified forms of antibodies, including but not limited tochimeric antibodies, humanized antibodies, heteroconjugate antibodies(e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies),single chain Fv antibodies (scFv), polypeptides that contain at least aportion of an immunoglobulin that is sufficient to confer specificantigen binding to the polypeptide, and antigen binding fragments ofantibodies. Antibody fragments include proteolytic antibody fragments[such as F(ab′)2 fragments, Fab′ fragments, Fab′-SH fragments, Fabfragments, Fv, and rIgG], recombinant antibody fragments (such as sFvfragments, dsFv fragments, bispecific sFv fragments, bispecific dsFvfragments, diabodies, and triabodies), complementarity determiningregion (CDR) fragments, camelid antibodies (see, for example, U.S. Pat.Nos. 6,015,695; 6,005,079; 5,874,541; 5,840,526; 5,800,988; and5,759,808), and antibodies produced by cartilaginous and bony fishes andisolated binding domains thereof (see, for example, International PatentApplication No. WO03014161).

A Fab fragment is a monovalent fragment consisting of the VL, VH, CL andCH1 domains; a F(ab′)₂ fragment is a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; an Fdfragment consists of the VH and CHI domains; an Fv fragment consists ofthe VL and VH domains of a single arm of an antibody; and a dAb fragmentconsists of a VH domain (see, e.g., Ward et al., Nature 341:544-546,1989). A single-chain antibody (scFv) is an antibody in which a VL andVH region are paired to form a monovalent molecule via a syntheticlinker that enables them to be made as a single protein chain (see,e.g., Bird et al., Science, 242: 423-426, 1988; Huston et al., Proc.Natl. Acad. Sci. USA, 85:5879-5883, 1988). Diabodies are bivalent,bispecific antibodies in which VH and VL domains are expressed on asingle polypeptide chain, but using a linker that is too short to allowfor pairing between the two domains on the same chain, thereby forcingthe domains to pair with complementary domains of another chain andcreating two antigen binding sites (see, e.g., Holliger et al., Proc.Natl. Acad. Sci. USA, 90:6444-6448, 1993; Poljak et al., Structure,2:1121-1123, 1994). A chimeric antibody is an antibody that contains oneor more regions from one antibody and one or more regions from one ormore other antibodies. An antibody may have one or more binding sites.If there is more than one binding site, the binding sites may beidentical to one another or may be different. For instance, a naturallyoccurring immunoglobulin has two identical binding sites, a single-chainantibody or Fab fragment has one binding site, while a “bispecific” or“bifunctional” antibody has two different binding sites.

In some examples, an antibody specifically binds to ERG or PTEN with abinding constant that is at least 10³ M⁻¹ greater, 10⁴ M⁻¹ greater or10⁵ M⁻¹ greater than a binding constant for other molecules in a sample.In some examples, a specific binding reagent (such as an antibody (e.g.,monoclonal antibody) or fragments thereof) has an equilibrium constant(K_(d)) of 1 nM or less. For example, a specific binding agent may bindto a target protein with a binding affinity of at least about 0.1×10⁻⁸M, at least about 0.3×10⁻⁸M, at least about 0.5×10⁻⁸M, at least about0.75×10⁻⁸ M, at least about 1.0×10⁻⁸M, at least about 1.3×10⁻⁸ Mat leastabout 1.5×10⁻⁸M, or at least about 2.0×10⁻⁸ M. Kd values can, forexample, be determined by competitive ELISA (enzyme-linked immunosorbentassay) or using a surface-plasmon resonance device such as the BiacoreT100, which is available from Biacore, Inc., Piscataway, N.J.

Methods of generating antibodies (such as monoclonal or polyclonalantibodies) are well established in the art (for example, see Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988). For example peptide fragments of ERG or PTEN (e.g.,fragments of SEQ ID NO: 2 or 4, respectively) can be conjugated tocarrier molecules (or nucleic acids encoding such epitopes or conjugatedRDPs) can be injected into non-human mammals (such as mice or rabbits),followed by boost injections, to produce an antibody response. Serumisolated from immunized animals may be isolated for the polyclonalantibodies contained therein, or spleens from immunized animals may beused for the production of hybridomas and monoclonal antibodies. In someexamples, antibodies are purified before use.

In one example, monoclonal antibody to ERG or PTEN (e.g., SEQ ID NO: 2or 4, respectively), can be prepared from murine hybridomas according tothe classical method of Kohler and Milstein (Nature, 256:495, 1975) orderivative methods thereof. Briefly, a mouse (such as Balb/c) isrepetitively inoculated with a few micrograms of the selected peptidefragment (e.g., epitope of ERG or PTEN) or carrier conjugate thereofover a period of a few weeks. The mouse is then sacrificed, and theantibody-producing cells of the spleen isolated. The spleen cells arefused by means of polyethylene glycol with mouse myeloma cells, and theexcess unfused cells destroyed by growth of the system on selectivemedia comprising aminopterin (HAT media). The successfully fused cellsare diluted and aliquots of the dilution placed in wells of a microtiterplate where growth of the culture is continued. Antibody-producingclones are identified by detection of antibody in the supernatant fluidof the wells by immunoassay procedures, such as ELISA, as originallydescribed by Engvall (Enzymol., 70:419, 1980), and derivative methodsthereof. Selected positive clones can be expanded and their monoclonalantibody product harvested for use.

Commercial sources of antibodies include Ventana Medical Systems (AZ),Epitomics (CA), Biocare (CA), Santa Cruz Biotechnology, Inc. (SantaCruz, Calif.), Cell Signaling Technology (Danvers, Mass.), and abcam(Cambridge, UK). Table 2 shows exemplary commercial sources ofantibodies for ERG and PTEN. A specific ERG antibody is theVentana/Epitomics Clone EPR 3864 and a specific PTEN antibody is theanti-PTEN rabbit monoclonal antibody (Cell Signaling Technology Clone138G6; catalog #9559).

TABLE 2 Exemplary commercial sources of antibodies. Antibody type SourceERG Rabbit Monoclonal Ventana (Epitomics EPR 3864). Mouse MonoclonalBiocare (clone 9F4) PTEN Monoclonal Santa Cruz Biotechnology, Inc.(sc-7974; sc-133197; sc-133242) Rabbit Monoclonal Cell SignalingTechnology (clone 138G6)

Disclosed specific binding agents also include aptamers. In one example,an aptamer is a single-stranded nucleic acid molecule (such as, DNA orRNA) that assumes a specific, sequence-dependent shape and binds to atarget protein (e.g., ERG or PTEN) with high affinity and specificity.Aptamers generally comprise fewer than 100 nucleotides, fewer than 75nucleotides, or fewer than 50 nucleotides (such as 10 to 95 nucleotides,25 to 80 nucleotides, 30 to 75 nucleotides, or 25 to 50 nucleotides). Ina specific embodiment, disclosed specific binding reagents aremirror-image aptamers (also called a SPIEGELMER™). Mirror-image aptamersare high-affinity L-enantiomeric nucleic acids (for example, L-ribose orL-2′-deoxyribose units) that display high resistance to enzymaticdegradation compared with D-oligonucleotides (such as, aptamers). Thetarget binding properties of aptamers and mirror-image aptamers aredesigned by an in vitro-selection process starting from a random pool ofoligonucleotides, as described for example, in Wlotzka et al., Proc.Natl. Acad. Sci. 99(13):8898-8902, 2002. Methods of generating aptamersare known in the art (see e.g., Fitzwater and Polisky (Methods Enzymol.,267:275-301, 1996; Murphy et al., Nucl. Acids Res. 31:e110, 2003).

In another example, an aptamer is a peptide aptamer that binds to atarget protein (e.g., ERG or PTEN) with high affinity and specificity.Peptide aptamers include a peptide loop (e.g., which is specific for thetarget protein) attached at both ends to a protein scaffold. This doublestructural constraint greatly increases the binding affinity of thepeptide aptamer to levels comparable to an antibody's (nanomolar range).The variable loop length is typically 8 to 20 amino acids (e.g., 8 to 12amino acids), and the scaffold may be any protein which is stable,soluble, small, and non-toxic (e.g., thioredoxin-A, stefin A triplemutant, green fluorescent protein, eglin C, and cellular transcriptionfactor Sp1). Peptide aptamer selection can be made using differentsystems, such as the yeast two-hybrid system (e.g., Gal4yeast-two-hybrid system) or the LexA interaction trap system.

Specific binding agents optionally can be directly labeled with adetectable moiety. Useful detection agents include fluorescent compounds(including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors, or the cyanine family of dyes (such as Cy-3 or Cy-5) and thelike); bioluminescent compounds (such as luciferase, green fluorescentprotein (GFP), or yellow fluorescent protein); enzymes that can producea detectable reaction product (such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, or glucose oxidaseand the like), or radiolabels (such as ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, or ¹³¹I).

4. Nucleic Acid Probes and Primers

In some examples, the means used to detect PTEN and ERG is a nucleicacid probe or primer. For example, nucleic acid probes or primersspecific for PTEN and ERG can be obtained from a commercially availablesource or prepared using techniques common in the art. Such agents canalso be used in the methods provided herein.

Nucleic acid probes and primers are nucleic acid molecules capable ofhybridizing with a target nucleic acid molecule (e.g., genomic targetnucleic acid molecule). For example, probes specific for a PTEN or ERGgene, when hybridized to the target, are capable of being detectedeither directly or indirectly. Primers specific for PTEN or ERG, whenhybridized to the target, are capable of amplifying the target gene, andthe resulting amplicons capable of being detected either directly orindirectly. Thus probes and primers permit the detection, and in someexamples quantification, of a target nucleic acid molecule, such as PTENand ERG.

Probes and primers can “hybridize” to a target nucleic acid sequence byforming base pairs with complementary regions of the target nucleic acidmolecule (e.g., DNA or RNA, such as genomic DNA, cDNA or mRNA), therebyforming a duplex molecule. Hybridization conditions resulting inparticular degrees of stringency will vary depending upon the nature ofthe hybridization method and the composition and length of thehybridizing nucleic acid sequences. Generally, the temperature ofhybridization and the ionic strength (such as the Na+ concentration) ofthe hybridization buffer will determine the stringency of hybridization.Calculations regarding hybridization conditions for attaining particulardegrees of stringency are discussed in Sambrook et al., (1989) MolecularCloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.(chapters 9 and 11). The following is an exemplary set of hybridizationconditions and is not limiting:

Very High Stringency (Detects Sequences that Share at Least 90%Identity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Detects Sequences that Share at Least 80% Identity)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Detects Sequences that Share at Least 50% Identity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

Commercial sources of probes and primers include Ventana MedicalSystems, AZ) and Vysis (IL). Table 3 shows exemplary PTEN and ERG primerpairs and Table 4 shows an exemplary CEN10 probe that can detect a PTENdeletion (if PTEN is intact 2 pairs of CEN10 and PTEN are expected onchromosome 10; if PTEN is heterozygous, 1 pair of PTEN/CEN10 and asingle CEN10 on the other copy of chromosome 10 is expected; if PTEN ishomozygous only 2 single CEN10 signals—one on each arm of chromosome10).

TABLE 3  Exemplary primers for ERG and PTEN. Target  Forward Primer Reverse Primer  Amp- gene (SEQ ID NO:) (SEQ ID NO:) licon ERG 5pTCCTTCCCCATCGGTTT ACGCAGAGATCAGTGAA 525 bp GTGGC(5) GGGAT (6) ERG 3pGTTTCTACACACGTTGC TCAAAAGGAATCACATT 107 bp CCACT(7) TACCACGGA (8) PTENTGACACCATGCAATCTT TGGGAAAGGATTGACAA 310 bp AAAAGCTGA (9) CTAAGAGGA (10)

TABLE 4  Exemplary probe for CEN10. CEN10GAATTCTTCTGTCTAGCAGTAAATGAGAAATCCCGCTTCCAA (pA10RP8 plasmid pool)CGAAGGCCTCAAACGGGTCTAACTAATCACTTGCAGACTTTACAGACAGAGTCTTTCCAAACTGCTCTATGAAGAGAAAGGTGAAACTCTGTGAACTGAACGCACAGATGACAAAGCAGTTTCTGAGAATGCTTCTGTGTAGTTTTTACACGAAGATATTTCCATTTCAAAGATTAGCCTCAAATCGCTTGAAATCTCCACTTGCAAACTCCACAGAAAGAATTTTTCAAAACTGCTCTGTCTAAAGGAAGGTTCAACTCTGTGACTTGAATACACACAACACAAAGAAGTGAC TGA

Methods of generating a probe or primer specific for a target nucleicacid (e.g., PTEN and ERG) are routine in the art (see e.g., Sambrook etal., (1989) Molecular Cloning, second edition, Cold Spring HarborLaboratory, Plainview, N.Y.). For example, probes and primers can begenerated that are specific for any of SEQ ID NOS: 1 or 3, such as aprobe or primer specific for at least 12, at least 50, at least 100, orat least 1000 contiguous nucleotides of such sequence (or itscomplementary strand). Probes and primers are generally at least 12nucleotides in length, such as at least 15, at least 18, at least 20, atleast 25, at least 30, at least 100, at least 1000, at least 5000, or atleast 6000 nucleotides, such as 12 to 100, 12 to 50, 12 to 30, 15 to 25,100 to 10,000, 1000 to 10,000 or 1000 to 6000 nucleotides. In oneexample, a primer for detecting ERG or PTEN is about 25 to 27 bp. In oneexample the probe is a FISH probe of at least 1000 bp, such as at least2000, at least 3000, at least 4000, at least 5000, or at least 6000,such as 1000 to 6000 bp, that covers from about 150 kb to 170 kb.Generally, probes include a detectable moiety or “label”. For example, aprobe can be coupled directly or indirectly to a “label,” which rendersthe probe detectable. In some examples, primers include a label thatbecomes incorporated into the resulting amplicon, thereby permittingdetection of the amplicon.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit a disclosed invention to the particular features or embodimentsdescribed.

Example 1 Increased ERG and Decreased PTEN Protein Expression Associatedwith Prostate Cancer Capsular Penetration

This example describes methods used to show that ERG over-expression andloss of PTEN expression is associated with capsular penetration inprostate cancer.

Prostate tissue samples from men were analyzed using IHC and antibodiesspecific for ERG and PTEN. Samples from 426 men who underwent openradical prostatectomy between 1997 and 2008 were obtained. The prostateswere totally embedded in paraffin. Only samples in which data forcapsular (CP) as well as non-capsular penetration (NCP) was available,were used for this analysis (see FIG. 1). Sections with no tumor tissuewere excluded from this analysis. Of the 426 men, 90 had capsularpenetration (CP) and 90 had pT3 lesions with an additional separatecontralateral lesion (non-capsular penetration, NCP, see FIG. 1). 18 menhave thus far shown biochemical recurrence. Capsular penetrating lesionsand the corresponding contralateral lesions were evaluated for ERGrearrangement and PTEN status by IHC. Normal PTEN expression (PTEN Pos)and absence of ERG (ERG Neg) were used as the reference group.

Formalin-fixed and paraffin-embedded (FFPE) samples analyzed usingimmunohistochemistry (IHC). ERG expression was analyzed using ananti-ERG rabbit monoclonal antibody (Ventana/Epitomics Clone EPR 3864)(FIG. 2). PTEN expression was analyzed using an anti-PTEN rabbitmonoclonal antibody (Cell Signaling Technology Clone 138G6; catalog#9559) with a goat anti-rabbit secondary antibody conjugated tohorseradish peroxidase (HRP, Ventana) and detected with the DABchromogen. Specificity of signals was evaluated visually by apathologist.

As shown in FIG. 3, prostate capsule penetration samples are PTEN− andERG+ in a representative sample. FIG. 4 shows a prostate capsulepenetration sample that is PTEN+ and ERG+ in a representative sample.FIG. 5 shows heterogeneous ERG expression in a prostate capsulepenetration sample. ERG expression was heterogeneous in 8/90 (9%) of CPand 7/90 (8%) of NCP lesions. FIG. 6 shows IHC results of ERG and PTENstaining. It was observed that invasive prostate cancers were associatedwith ERG positive high grade prostatic intraepithelial neoplasia (HGPIN)lesions in 21/90 (23.3%) of CP and 22/90 (24.4%) of NCP. This resultindicates that ERG deletion and over-expression alone is unlikely to beprognostic of aggressive outcomes in the assessment of prostate cancer.All normal glands showed robust cytoplasmic staining for PTEN. Negativestaining corresponding to lack of PTEN expression was observed in 31 of90 (34%) of the capsular penetrating carcinoma lesions and 18 of 90(20%) of the contralateral organ confined carcinoma lesions. Thecarcinoma lesions revealed heterogeneity of PTEN expression in 15 of 90(16.6%) of the capsular penetrating carcinoma lesions and 11 of 90(12.2%) of the contralateral organ confined carcinoma lesions.

Heterogeneity of the prostate cancer indicates that individual glandsneed to be assessed for the molecular rearrangements (example in ERG)and/or deletions (example in PTEN), including over-expression (examplein ERG) and/or loss of expression (example in PTEN), or changes inmorphology of the nuclei for the disease prognosis to be established.

If only ERG staining was analyzed in the absence of PTEN, there was nosignificant difference in the percentage of positive cases between thecapsular penetration tumors and the non-capsular penetration tumors(p=0.54). There was no significant difference in the percentage of ERGpositive tumors in the lesions with Gleason sum score ≧7 and <7.

As shown in Table 5, an odds ratio (OR) of 4.88 was observed for samplesnegative for PTEN protein expression and having increased ERG expression(indicating that capsular penetration is about 4.88-times more likely inlesions negative for PTEN expression and positive for ERGover-expression) than the normal reference (ERG−; PTEN+). This profilewas generated after comparison with prostate cancer lesionsdemonstrating a normal expression profile for PTEN and ERG (Pos PTEN andNeg ERG). Based on these results, having an expression profile of PTENloss and ERG over-expression is a risk factor for capsular penetrationin prostate cancer.

TABLE 5 Association with Capsular Penetration Non- Capsular CapsularPenetrated Penetrated Profile Lesion Lesion OR (95% CI) p-valueERG−/PTEN+ 42 (47%) 44 (49%) refer- ence ERG+/PTEN+ 29 (32%) 36 (40%)0.84 (0.42, 1.69) 0.61 ERG+/PTEN− 14 (16%) 3 (3%) 4.88 (1.22, 28.0) 0.01ERG−/PTEN− 5 (6%) 7 (8%) 0.75 (0.17, 2.99) 0.64 Total  90 (100%)  90(100%)

Table 6 shows the association with Gleason sum score. Positive PTEN isassociated with statistically significant lower Gleason sum score.

TABLE 6 Marker status by Gleason score. Gleason sum scores, N (%) MarkerStatus 6 7 8 10 Total p-value ERG Negative 22 13 12 0  47 0.65 (46.1)(27.7) (25.5) (100) Positive 22 12 8 1  43 (51.2) (27.9) (18.6) (2.3)(100) PTEN Negative 3 7 8 1  19 0.003 (15.8) (36.8) (42.1) (5.3) (100)Positive 41 18 12 0  71 (57.8) (25.4) (16.9) (100)

FIG. 7 shows Kaplan-Meier plots showing estimates for biochemicalrecurrence by ERG/PTEN profile of the capsular penetrated lesion. Asshown in FIG. 7, PTEN− prostate cancers (such as those that are ERG+ orERG−) are very highly aggressive, as indicated by increased incidencesof biochemical recurrence (such as 1, 3 or 5 years post-prostatectomy).For example, at 5-years post-prostatectomy, patients with ERG+/PTEN+ orERG−/PTEN+ tumors had a lower rate of biochemical recurrence thanpatients with ERG−/PTEN− or ERG+/PTEN− tumors (which have a higher rateof biochemical recurrence).

Example 2 Increased ERG and Decreased PTEN Genomic Expression Associatedwith Prostate Cancer Capsular Penetration

A subset of recurrent cases was subjected to four color quantum dot FISHfor evaluation of ERG gene rearrangement and PTEN gene deletions.

ERG gene rearrangements and PTEN deletions were detected in asimultaneous four probe/four Quantum Dot FISH assay (Benchmark®XT,Ventana) (FIGS. 8 and 9), essentially as described in Svensson et al.(Lab. Invest. 91:404-12, 2011, herein incorporated by reference) withthe exception that FISH probes for 3p and 5′ERG were used together withthose for PTEN and CEN10. ERG probe locations have been described(Tomlins et al. Science 310(5748):644-8, 2005, herein incorporated byreference). PTEN FISH probes are located on 10q23.31 and were derivedfrom BAC clones RP11-659F22, RP11-210E13, CTD-2557P6, CTD-3243P1, andRP11-765C10 (LifeTechnologies, OR). The CEN10 (centromere 10) probe isinserted in a DNA plasmid pA10RP8 (ATCC, VA). All probes were purifiedand labeled using nick translation. Specifically, 5p ERG was labeledwith dUTP DIG (digoxigenin, Roche), 3pERG with dCTP DNP (dinotrophenyl,Ventana), PTEN with dUTP TS (thiazole sulphonamide, Ventana), and CEN10with dUTP NP (nitropyrazole, Ventana).

Detection was conducted online as a part of the automated protocol withmonoclonals to DIG (mouse mAb, Roche), DNP (rat mAb LO-DNP, U Louvain,Brussels), TS (clone #13A06-01E11, Ventana) and NP (clone #806263,Ventana) conjugated to Quantum Dot 565, Quantum Dot 655, Quantum Dot605, and Quantum Dot 585 respectively. All antibody conjugations areconducted using 30n PEGylated Quantum dots (Life Technologies, OR) andpurified monoclonal antibodies. Quantum Dot-DAPI (Ventana) was appliedonline to counter-stain nuclei for evaluation and imaging. Regions ofinterest were selected in DAPI stained nuclei with 30 to 100 nucleievaluated per case.

Concordance between FISH and IHC methods was observed. Of the 22 FISHsamples there were 3 ERG and 6 PTEN tests which were non-informative. Ofthe 19 informative ERG FISH tests, 16 (84%) were concordant with the IHCresult. Of the 6 ERG rearranged cases, 5 were deleted and 1 was breakapart. Of the 16 informative PTEN FISH results 14 (88%) were concordantwith the IHC result.

In summary, the lack of PTEN expression is associated with increasedrisk of capsular penetration, and men with an ERG+/PTEN− tumor have ahigher risk of prostate cancer capsular penetration and earlierbiochemical recurrence of prostate cancer.

Example 3 In Situ Hybridization to Detect Expression

This example provides exemplary methods that can be used to detect geneexpression using in situ hybridization, such as FISH or CISH. Althoughparticular materials and methods are provided, one skilled in the artwill appreciate that variations can be made.

Prostate cancer tissue samples (e.g., samples that have penetrated thecapsule), such as FFPE samples, are mounted onto a microscope slide,under conditions that permit detection of nucleic acid molecules presentin the sample. For example, cDNA or mRNA in the sample can be detected.The slide is incubated with nucleic acid probes that are of sufficientcomplementarity to hybridize to cDNA or mRNA in the sample under veryhigh or high stringency conditions. Probes can be RNA or DNA. Separateprobes that are specific for ERG and PTEN nucleic acid sequences (e.g.,human sequences) are incubated with the sample simultaneously orsequentially, or incubated with serial sections of the sample. Forexample, each probe can include a different fluorophore or chromogen topermit differentiation between the three probes. After contacting theprobe with the sample under conditions that permit hybridization of theprobe to its gene target, unhybridized probe is removed (e.g., washedaway), and the remaining signal detected, for example using microscopy.In some examples, the signal is quantified.

In some examples, additional probes are used, for example to detectexpression of one or more other prostate cancer related genes or one ormore control genes (e.g., β-actin). In some examples, expression of ERGand PTEN is also detected (using the same probes) in a control sample,such as a breast cancer cell, a prostate cancer cell from a subject whodoes not have an aggressive prostate cancer that has penetrated theprostatic capsule, a prostate cancer cell from a subject who has anaggressive prostate cancer that has penetrated the prostatic capsule, anon-cancer cell adjacent to the tumor, or a normal (non-cancer) cell.

The resulting hybridization signals for ERG and PTEN are compared to acontrol, such as a value representing ERG and PTEN expression in anormal (non-cancerous) prostate sample, such as a prostate sample thatis ERG−/PTEN+. If increased expression of ERG, and decreased expressionof PTEN, relative to a value representing ERG and PTEN expression in anormal prostate sample, this indicates that the subject has a poorprognosis (e.g., less than a 1 or 2 year survival) as the cancer islikely to recur. Similarly, if ERG and PTEN expression are similarrelative to a value representing ERG and PTEN expression in a prostatecancer cell from a subject who has an aggressive prostate cancer thathas penetrated the prostatic capsule (e.g., an ERG+/PTEN− prostatecancer sample), this indicates that the subject has a poor prognosis(e.g., the cancer is likely to recur, metastasize, and/or the patienthas a shorter life expectancy). If ERG and PTEN expression are similar(e.g., no more than a 2-fold difference) relative to a valuerepresenting ERG and PTEN expression in a prostate cancer sample from asubject who does not have an aggressive prostate cancer that haspenetrated the prostatic capsule, this indicates that the subject has agood prognosis (e.g., the cancer is not likely to recur, notmetastasize, and/or the patient has a longer life expectancy).

Example 4 Nucleic Acid Amplification to Detect Expression

This example provides exemplary methods that can be used to detect geneexpression using nucleic acid amplification methods, such as PCR.Amplification of target nucleic acid molecules in a sample can permitdetection of the resulting amplicons, and thus detection of expressionof the target nucleic acid molecules. Although particular materials andmethods are provided, one skilled in the art will appreciate thatvariations can be made.

RNA is extracted from a prostate cancer tissue sample (such as one thathas penetrated the capsule), such as FFPE samples or fresh tissuesamples (e.g., surgical specimens). Methods of extracting RNA areroutine in the art, and exemplary methods are provided elsewhere in thedisclosure. For example RNA can be extracted using a commerciallyavailable kit. The resulting RNA can be analyzed as described in Example1 to determine if it is of an appropriate quality and quantity.

The resulting RNA can be used to generate DNA, for example using RT-PCR,such as qRT-PCR. Methods of performing PCT are routine in the art. Forexample, the RNA is incubated with a pair of oligonucleotide primersspecific for the target gene (e.g., ERG and PTEN). Such primers are ofsufficient complementarity to hybridize to the RNA under very high orhigh stringency conditions. Primer pairs specific for ERG and PTENnucleic acid sequences (e.g., human sequences) can be incubated withseparate RNA samples (e.g., three separate PCR reactions are performed),or a plurality of primer pairs can be incubated with a single sample(for example if the primer pairs are differentially labeled to permit adiscrimination between the amplicons generated from each primer pair).For example, each primer pair can include a different fluorophore topermit differentiation between the amplicons. Amplicons can be detectedin real time, or can be detected following the amplification reaction.Amplicons are usually detected by detecting a label associated with theamplicon, for example using spectroscopy. In some examples, the ampliconsignal is quantified.

In some examples, additional primer pairs are used, for example todetect expression of one or more other prostate cancer related genes, orone or more control genes (e.g., β-actin). In some examples, expressionof ERG and PTEN is also detected (using the same probes) in a controlsample, such as a breast cancer cell, a prostate cancer cell from asubject who does not have an aggressive prostate cancer that haspenetrated the prostatic capsule, a prostate cancer cell from a subjectwho has an aggressive prostate cancer that has penetrated the prostaticcapsule, a non-cancer cell adjacent to the tumor, or a normal(non-cancer) cell.

The resulting amplicon signals for ERG and PTEN are compared to acontrol, such as a value or range of values representing ERG and PTENexpression in a normal (non-cancerous) sample (e.g., a sample that isERG−/PTEN+). If increased expression of ERG, and decreased expression ofPTEN, relative to a value representing ERG and PTEN expression in anormal sample, this indicates that the subject has a poor prognosis(e.g., less than a 1 or 2 year survival) as the cancer is likely torecur. Similarly, if ERG and PTEN expression are similar relative to avalue representing ERG and PTEN prostate cancer cell from a subject whohas an aggressive prostate cancer that has penetrated the prostaticcapsule, this indicates that the subject has a poor prognosis (e.g., thecancer is likely to recur, metastasize, and/or the patient has a shorterlife expectancy). If ERG and PTEN expression are similar (e.g., no morethan a 2-fold difference) relative to a value representing ERG and PTENexpression in a prostate cancer sample from a subject who does not havean aggressive prostate cancer that has penetrated the prostatic capsule,this indicates that the subject has a good prognosis (e.g., the canceris not likely to recur, not metastasize, and/or the patient has a longerlife expectancy).

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples of the disclosure and should not be takenas limiting the scope of the invention. Rather, the scope of thedisclosure is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

We claim:
 1. A method for determining risk that a prostate cancer willpenetrate the prostatic capsule, comprising: contacting a prostatecancer sample isolated from a subject with an ETS related gene(ERG)-specific antibody and a phosphatase and tensin homolog(PTEN)-specific antibody; measuring increased protein expression of ERGin the prostate cancer sample relative to a control representing ERGprotein expression expected in a normal prostate sample; measuringdecreased PTEN in the prostate cancer sample relative to a controlrepresenting PTEN protein expression expected in a normal prostatesample; determining that there is a higher risk that the prostate cancerhas penetrated or will likely penetrate the prostatic capsule based onthe ERG measuring and the PTEN measuring; and administering atherapeutic agent for treating prostate cancer to the subject from whichthe prostate cancer sample was obtained, performing a prostatectomy onthe subject from which the prostate cancer sample was obtained, orcombinations thereof.
 2. The method of claim 1, wherein the prostatecancer sample is a prostate cancer sample that has not penetrated theprostatic capsule, and the method determines that the risk that theprostate cancer will penetrate the prostatic capsule in the future is atleast four-times more likely when increased expression of ERG anddecreased expression of PTEN is measured in the prostate cancer samplerelative to the control.
 3. The method of claim 1, wherein it is notknown whether the prostate cancer sample has penetrated the prostaticcapsule prior to performing the method, and the method determines thatthe cancer has penetrated the prostatic capsule when increasedexpression of ERG and decreased expression of PTEN is measured in theprostate cancer sample relative to the control.
 4. The method of claim1, further comprising: measuring expression of one or more otherprostate cancer-related molecules in the sample; and comparingexpression of the one or more other prostate cancer related molecules inthe prostate cancer sample to a control representing expression of theone or more other prostate cancer-related molecules expected in a normalprostate sample.
 5. The method of claim 1, wherein the prostate cancersample is a fixed, wax-embedded prostate cancer tissue sample.
 6. Themethod of claim 1, wherein the prostate cancer sample is collected afterprostate cancer diagnosis and after prostatectomy in the subject.
 7. Themethod of claim 1, wherein the prostate cancer sample is collected fromtissue removed during a prostatectomy.
 8. The method of claim 1, whereinthe ERG-specific antibody is a rabbit monoclonal antibody produced fromhybridoma clone EPR 3864 and the PTEN-specific antibody is a rabbitmonoclonal antibody produced from hybridoma clone 138G6.
 9. The methodof claim 1, further comprising generating a report, wherein the reportcomprises a risk that a prostate cancer will penetrate the prostaticcapsule.
 10. The method of claim 1, wherein the therapeutic agent isradiation, a chemotherapeutic, or a hormone.
 11. The method of claim 10,wherein the chemotherapeutic comprises temozolomide or docetaxel.